Two stage nucleic acid amplification using an amplification oligomer

ABSTRACT

This invention provides methods, compositions and systems to detect a nucleic acid of interest in a two-stage amplification. The two-stage amplification begins with a first non-enzymatic accumulation of an amplification oligomer that is the target substrate for a second nucleic acid amplification or assay. Two or more amplification oligomers can be used to allow multiplexed amplifications of two or more nucleic acids of interest with deconvolution based on unique detection signals or unique signal locations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application claiming priority to andbenefit of a prior to parent application Ser. No. 11/983,216, Two-StageNucleic Acid Amplification Using an Amplification Oligomer, by GaryMcMaster, filed Nov. 6, 2007, which claims priority to and benefit of aprior U.S. Provisional Application No. 60/872,199, Two-Stage NucleicAcid Amplification Using an Amplification Oligomer, by Gary McMaster, etal., filed Dec. 1, 2006. The full disclosure of the prior application isincorporated herein by reference.

FIELD OF THE INVENTION

The inventions are in the field of specific nucleic acid amplificationand detection. The present methods and systems include a first targetnucleic acid capture that provides, e.g., a first non-enzymaticamplification accumulating amplification oligomers that can become thetarget substrate for a second nucleic acid amplification or nucleic acidassay. Unique sequences in two or more different amplification oligomerscan be used to direct them to be captured at separate locations or tocapture reporter molecules with different signals, thus allowinganalyses of two or more different nucleic acids at once from the samesample.

BACKGROUND OF THE INVENTION

A wide variety of techniques for amplifying nucleic acids are known inthe art, including, but not limited to, PCR (polymerase chain reaction),rolling circle amplification, and transcription mediated amplification.(See, e.g., Hatch et al. (1999) “Rolling circle amplification of DNAimmobilized on solid surfaces and its application to multiplex mutationdetection” Genet Anal. 15:35-40; Baner et al. (1998) “Signalamplification of padlock probes by rolling circle replication” NucleicAcids Res. 26:5073-8; and Nallur et al. (2001) “Signal amplification byrolling circle amplification on DNA microarrays” Nucleic Acids Res.29:E118.) A labeled primer and/or labeled nucleotides are optionallyincorporated during amplification. In many embodiments, the nucleicacids of interest are captured and amplified or detected but without anability distinguish between two or more nucleic acids from the samesample. Further, the enzyme dependent amplification techniques often runinconsistently depending on the purity and complexity of the samplesprovided.

PCR amplifications are commonly used in nucleic acid analysis ofsamples, but suffer from limited amplicon size and difficultiesproviding conditions for consistent enzymatic activity. These problemsare only heightened in analyses requiring reliable quantitation. Forexample, performance of quantitative PCR (QPCR) has faired poorly inquantitation of complex or degraded samples because it is generallylimited to 75-85 by amplicon size, and multiple pooled gene-specificprimers are required. QPCR requires a much greater nucleic acid puritythan the bDNA assay and thus more steps to process the samples prior toanalysis compared to the bDNA technology. A second problem that affectsRNA quantification by QPCR is the required reverse transcription step toconcert mRNA sequences of interest to cDNA. This enzymatic reaction isimpeded by any base modifications, by secondary mRNA structure and byimpurities in the RNA preparation. Although, introduction of a hightemperature heating step during PCR amplification steps may partiallyreverse some of the RNA base modifications, for many samples thesemodifications are irreversible. Older samples are often so impaired thata decrease in average QPCR signal is >90%, requiring more input RNA andincreasing Ct values to 35-40 (Masuda N, Ohnishi T, Kawamoto S, MondenM, Okubo K: Analysis of chemical modification of RNA from formalin-fixedsamples and optimization of molecular biology applications for suchsamples, Nucleic Acids Res 1999, 27:4436-4443). With all these problems,QPCR has not been a satisfactory method of quantitating many types ofnucleic acid samples. Furthermore these problems can not be cured bycontinued PCR amplification of the sample. Input of PCR products of afirst amplification into a second full series of PCR amplifications onlytends to further compound the amplification of errors originating in thefirst amplification.

One method of DNA amplification has the distinct advantage of not beingdependent on enzymes to generate a signal. In a typical bDNA assay forgene expression analysis, a target mRNA whose expression is to bedetected is released from cells and captured by a capture probe (CP) ona solid surface (e.g., a well of a microtiter plate) through syntheticoligonucleotide probes called capture extenders (CEs). Each captureextender has a first polynucleotide sequence that can hybridize to thetarget mRNA and a second polynucleotide sequence that can hybridize tothe capture probe. Typically, two or more capture extenders are used.Probes of another type, called label extenders (LEs), hybridize todifferent sequences on the target mRNA and to sequences on anamplification multimer. Additionally, blocking probes (BPs), whichhybridize to regions of the target mRNA not occupied by CEs or LEs, areoften used to reduce non-specific target/probe binding. A probe set fora given mRNA thus consists of CEs, LEs, and optionally BPs for thetarget mRNA. The CEs, LEs, and BPs are complementary to nonoverlappingsequences in the target mRNA, and are typically, but not necessarily,contiguous. Signal amplification begins with the binding of the LEs tothe target mRNA. An amplification multimer is then typically hybridizedto the LEs. The amplification multimer has multiple copies of a sequencethat is complementary to a label probe (it is worth noting that theamplification multimer is typically, but not necessarily, abranched-chain nucleic acid; for example, the amplification multimer canbe a branched, forked, or comb-like nucleic acid or a linear nucleicacid). A label, for example, alkaline phosphatase, is covalentlyattached to each label probe. (Alternatively, the label can benon-covalently bound to the label probes.) In the final step, labeledcomplexes are detected, e.g., by the alkaline phosphatase-mediateddegradation of a chemilumigenic substrate, e.g., dioxetane. Luminescenceis reported as relative light unit (RLUs) on a microplate reader. Theamount of chemiluminescence is proportional to the level of mRNAexpressed from the target gene.

An exemplary embodiment of bDNA technology is schematically illustratedin FIG. 1, wherein a single target nucleic acid is captured and detectedas an accumulation of label probes. A cell or tissue sample is lysed toproduce a lysate including target nucleic acid 114. The target nucleicacid 114 (e.g., an mRNA whose expression is to be detected) is capturedby capture probe 104 on solid support 101 (e.g., a well of a microtiterplate) through set 111 of synthetic oligonucleotide capture extenders.Each capture extender has a first polynucleotide sequence C-3 (152) thatcan hybridize to the target nucleic acid and second polynucleotidesequence C-1 (151) that can hybridize to the capture probe throughsequence C-2 (150) in the capture probe. Typically, two or more captureextenders are used; optionally, one CE can be used to capture a target.Each label extender in label extenders set 121 hybridizes to a differentsequence on the target nucleic acid, through sequence L-1 (154) that iscomplementary to the target nucleic acid, and to sequence M-1 (157) onamplification multimer (141), through sequence L-2 (155). Blockingprobes (124), which hybridize to sequences in the target nucleic acidnot bound by either capture extenders or label extenders, are often usedin bDNA assays to reduce non-specific target probe binding. A probe setfor a given target nucleic acid thus consists of capture extenders,label extenders, and optional blocking probes 124 for the target nucleicacid. The capture extenders, label extenders, and optional blockingprobes are complementary to non-overlapping sequences in the targetnucleic acid, and are typically, but not necessarily, contiguous. Inthis example, a single blocking probe is used; typically, an array ofdifferent blocking probes is used in an optimized bDNA assay.

Signal amplification can begin with the binding of the label extendersto the target nucleic acid. The amplification multimer is thenhybridized to the label extenders. The amplification multimer hasmultiple copies of sequence M-2 (158) that is complementary to labelprobe 142. Label 143, for example, a fluorescent group, is covalentlyattached to each label probe. In the final step, labeled complexes aredetected, e.g., by fluorometry. The amount of fluorescence can beproportional to the level of target nucleic acid originally present inthe sample (a relationship describable, e.g., by a regression curve).However, the amplifications of a bDNA assay are limited, e.g., by thenumber of label probe sequence sites available on the amplificationmultimer and stearic hindrance in the amplification complex. Whendetecting two or more different target nucleic acids from the samesample, the typical bDNA assay provides only a combined result withoutseparate identification or quantitation.

In view of the above, a need exists for methods of amplifying nucleicacids to a higher degree. It would be desirable to have systems that canhighly amplify signals associated with the presence of a particularnucleic acid of interest in a sample without the use of amplifyingenzymes. Benefits can be obtained from methods and systems capable ofamplifying, quantitating and uniquely identifying signals from two ormore different nucleic acid targets in the same assay. The presentinvention provides these and other features that will be apparent uponreview of the following.

SUMMARY OF THE INVENTION

The present invention uses the unique characteristics of the two-stageamplification schemes described herein to provide powerful methods andsystems to detect, quantify and/or identify one or more nucleic acids ofinterest in a sample. The methods include, e.g., a first bDNAamplification to provide a first amplification product, an amplificationoligomer, that can act as a substrate for a second amplification ornucleic acid assay. The sequences of amplification oligomers can includeencoded information useful in, e.g., associating the oligomer with thetest target nucleic acid, directing a signal to a particular physicallocation, providing a substrate for an amplification enzyme, providing atarget for a second bDNA amplification, and/or selecting a uniquedistinguishable reporter from an assay solution. The two-stageamplification schemes of the invention can drastically increase thesensitivity of a nucleic acid assay, identify the presence of a rarenucleic acid in a complex mixture of nucleic acids, determine thequantity of a rare or dilute nucleic acid in a sample, and/or allowquantitative and qualitative assays distinguishing two or more nucleicacids of interest from the same sample at once.

In a preferred embodiment of detecting a target nucleic acid of interestin a sample, each target is amplified to provide a number ofamplification oligomers in a first non-enzymatic amplification, followedby a second amplification of the amplification oligomers to provide astill larger number of readily detectable labeled probes. For example,the method of detecting a nucleic acid of interest can include providinga sample comprising or suspected of comprising one or more nucleic acidsof interest, capturing those nucleic acids of interest present in thesample on a first solid support, hybridizing a first amplificationmultimer directly or indirectly to the nucleic acid of interest,hybridizing the amplification multimer to one or more amplificationoligomers, and detecting the previously hybridized amplificationoligomers in a non-enzymatic nucleic acid assay. In a preferredembodiment, the non-enzymatic nucleic acid assay includes the steps ofwashing unbound components of the first amplification from the firstsolid support, melting the bound amplification oligomers from the firstsolid support and transferring them to a second solid support. Thetransferred amplification oligomers are then captured, e.g., by captureprobes having sequences complimentary to the amplification oligomers onthe second solid support. Second amplification multimers are hybridizeddirectly or indirectly to the complimentary sequences on theamplification oligomers (now acting as a target nucleic acid) and labelprobes are hybridized onto multiple complimentary sequences on theamplification multimer to provide abundant label signal. The presence ofthe target nucleic acid of interest in the original sample can bedetermined based on the amount of signal from label probe detected atthe second solid support.

The amplification oligomers of the invention can have various sequencesuseful in directing them to bind at a solid support and useful in theiramplification and detection. For example, amplification oligomers of theinvention can include a nucleotide sequence complimentary to anamplification multimer M-2 sequence of a first amplification multimer, anucleotide sequence complimentary to a capture extender C-3 sequence orto a capture probe C-2 sequence, and a nucleotide sequence complimentaryto a label extender L-1 sequence or to an amplification multimer M-1sequence of a second amplification multimer. In a preferred embodiment,the first amplification multimer is a component of a first bDNA assay,and the capture extender or the capture probe are components of a secondbDNA assay. In preferred embodiments, the amplification oligomernucleotide sequence complimentary to the amplification multimer M-2sequence comprises from 20 to 80 nucleotides, or from 30 to 60nucleotides, or about 50 nucleotides. In a preferred embodiment, thenucleotide sequence of the amplification oligomer complimentary to thecapture extender C-3 sequence or to the capture probe C-2 sequencecomprises from 20 to 60 nucleotides or about 50 nucleotides. In stillmore preferred embodiments, amplification oligomer nucleotide sequencecomplimentary to the label extender L-1 sequence or to the amplificationmultimer M-1 sequence of the second amplification multimer comprisesfrom 20 to 40 nucleotides or about 28 nucleotides. In a typicalembodiment, the amplification oligomer comprises a total length from 60to 300 nucleotides, from 100 to 150 nucleotides, or about 130nucleotides. In preferred embodiments, the amplification oligomers donot comprise a label.

The amplification oligomers can have complimentary sequences in theorder: 1) the amplification multimer M-2 compliment; 2) the captureextender compliment or capture probe compliment; 3) label extendercompliment or amplification M-1 compliment. In optional embodiments, theamplification oligomer does not include a compliment to a capturesystem. In an alternate preferred embodiment, the amplification oligomernucleotide sequence complimentary to the amplification multimer M-2sequence of a first amplification multimer is between 1) the nucleotidesequence complimentary to a capture extender C-3 sequence or to acapture probe C-2 sequence of a second amplification, and the nucleotidesequence complimentary to the label extender L-1 sequence or to anamplification multimer M-1 sequence of a second amplification multimer.

In preferred embodiments, particularly in matrixed amplificationembodiments, the amplification oligomer has two or more sequencescomplimentary to a capture extender C-3 sequence or to a capture probeC-2 sequence, thus allowing cooperative hybridization in a secondcapture step. In a similar fashion, it is preferred that theamplification oligomer have two or more sequences complimentary to thelabel extender L-1 sequence or to the amplification multimer M-1sequence of the second amplification multimer to allow the benefits ofcooperative hybridization.

In the two-stage amplification methods, the target nucleic acid ofinterest (or amplification oligomer in a second non-enzymaticamplification) can be captured on a solid support directly, indirectly,covalently, by affinity, by hybridization, etc. For example, capturingcan be through hybridization of a first capture probe on the solidsupport to a first capture extender and hybridization of the captureextender to a complimentary sequence on the nucleic acid of interest;through hybridization of the nucleic acid of interest directly to acapture probe; by affinity capture of the nucleic acid of interest(e.g., antibody capture of a hapten on an incorporated nucleotideanalog); by capture of the nucleic acid of interest by the solid supportsubstrate (e.g., by formation of a covalent bond in a chemical reactionor by molecular interactions, such as ionic, chelation or hydrophobicinteractions).

The label probe system used to detect the presence of a nucleic acid ofinterest can be configured in a variety of ways. For example, the labelprobe system can include a covalently or non-covalently branchedstructure decorated with label probes and associated with theamplification oligomer in a second non-enzymatic amplification through alabel extender. That is, the label system can include a label probe witha sequence complimentary to an amplification multimer, which hassequences complimentary to a label extender, which has sequencescomplimentary to the amplification oligomers. The label probe system canbe configured to hybridize the amplification multimer directly to theamplification oligomer in the second amplification, e.g., anamplification multimer with sequences directly complimentary to theamplification oligomer, and to multiple label probes. Optionally, the alabel probe system can include a system wherein the amplificationoligomer consists of preamplifiers and amplifier nucleic acid strands,e.g., a preamplifier with a sequence complimentary to the amplificationoligomer (optionally, indirectly through a label extender) and to anamplifier with a sequence comprising multiple sequence sitescomplimentary to label probes.

Noise can be reduced in the amplifications and sensitivity increased byreducing non-specific binding and careful control of hybridizationstringency. In preferred embodiments, the label extender and captureextender are present in excess over complimentary first amplificationmultimer sequences in the second amplification. Blocking oligomerscomplimentary to the amplification oligomer can be provided in the firstand/or second amplification reactions to reduce hybridization ofamplification oligomers to first amplification multimers. In manyembodiments, blocking probes complimentary to target nucleic acidstrands not complimentary to intended label systems or capture systemsare provided to reduce non-specific binding of undesired sample orsystem nucleic acids.

The nucleic acid of interest captured in the non-enzymaticamplifications of the invention can be nucleic acids from clinicalspecimens, research samples, forensic samples, and the like. The nucleicacids can be any type, such as e.g., DNA, cDNA, RNA, mRNA, rRNA, miRNA,siRNA and/or the like. In many embodiments, the target nucleic acid ofinterest is the amplification product of the first amplification, e.g.,an amplification oligomer. The product of a first amplification can bedetected in a nucleic acid assay, such as, e.g., a bDNA assay, PCR, LCR,a northern blot, a Southern blot, electrophoresis, and light absorbance.A positive result in the assay can be correlated to the presence of anucleic acid of interest in the test sample.

Ultimate detection of a signal from the second amplification can be byany appropriate detection technique. For example, detection of thesecond amplification product can be by a bDNA assay, a northern blot, aSouthern blot, electrophoresis, and light absorbance. In manyembodiments, labeled product results from the second amplification,allowing ready detection of the product, e.g., by fluorometry,spectrophotometry, phosphorimaging, etc. For example, where the secondamplification is a bDNA assay, a large amount of label probes, e.g.,with fluorescent labels, can be present on amplification multimers at asolid support associated with a positive result for the presence of anucleic acid of interest. In another example, wherein the secondamplification is a PCR assay, the replicated nucleic acid canincorporate labeled nucleotides or be detectable using FRET (e.g.,Taqman) probes.

Methods of the invention further provide a variety of ways to multiplextwo or more nucleic acids of interest through the two-stageamplifications so that their presence can each be separately detected.The presence or absence of two or more nucleic acids of interest can bedetermined, e.g., by providing a sample comprising or suspected ofcomprising one or more nucleic acids of interest, capturing thosenucleic acids of interest present in the sample on a first solidsupport, providing a first amplification multimer comprising a first M-1sequence and capable of hybridizing directly or indirectly to a firstnucleic acid of interest, providing a second amplification multimercomprising a second M-1 sequence different from the first M-1 sequenceand capable of hybridizing directly or indirectly to a second nucleicacid of interest different from the first nucleic acid of interest;providing a first amplification oligomer comprising a sequencecomplimentary to the first amplification multimer and a secondamplification oligomer comprising a sequence complimentary to the secondamplification multimer, directly or indirectly hybridizing the capturednucleic acids of interest to the first or second amplificationmultimers, hybridizing the amplification oligomers to the hybridizedamplification multimers, and detecting the previously hybridizedamplification oligomers in a nucleic acid assay. Depending on the natureof the nucleic acid assay, the presence and/or quantity of each nucleicacid of interest can be determined from the same sample.

Different amplification oligomers accumulated on the same first solidsupport in a matrixed assay can be washed to remove residual componentsof the first amplification and then melted in a melting solution fortransfer to one or more separate solid supports for one or moreadditional amplifications. For example, the amplification oligomers canbe captured on a second solid support by, e.g., hybridizing a captureprobe on the second solid support to a capture extender and hybridizingthe capture extender to a complimentary sequence on the amplificationoligomer; hybridizing the amplification oligomer directly to a captureprobe on the second solid support; affinity capture of the amplificationoligomer, and/or capture of the amplification oligomer through chemicalinteractions with the solid support substrate. The amplificationoligomers captured on a second solid support can be detected, e.g.,hybridizing the amplification oligomers to a preamplifier, hybridizingthe preamplifier to an amplifier, hybridizing a label probe to theamplifier, and detecting a signal associated with the label probe. Thedetections of two or more different amplification oligomers can takeplace at the same locations on the additional solid support or atdifferent locations. For example, the first amplification oligomers canbe captured on a first of the additional solid supports and the secondamplification oligomers can bee captured on a second of the additionalsolid supports, so that the presence of the first nucleic acid ofinterest can be detected as a signal emanating from the first solidsupport or the presence of the second nucleic acid of interest can bedetected as a signal emanating from the second solid support.

In preferred embodiments of matrixed amplifications, the nucleic acidassay after the first amplification can be a second amplification, e.g.,a second non-enzymatic amplification. For example, a first amplificationoligomer product of the first amplification can have a sequencecomplimentary to a capture extender or capture probe on a second solidsupport, and a different second amplification oligomer product of thefirst amplification can have a sequence complimentary to a differentcapture extender or capture probe on the second solid support. Thelocations of the different capture probes on the solid support can beseparated or common. For example, the capture extender or capture probecomplimentary to the first amplification oligomer can be bound to thesecond solid support at one location and the other capture extender orcapture probe complimentary to the second amplification oligomer can bebound to the second solid support at a different location. Optionally,the first and second amplification oligomers can be captured at entirelydifferent solid supports (e.g., different beads). In such a case,detecting second amplification products can include contacting theamplification oligomers in a solution to second and third solid supportsat the same time or contacting them in sequence.

The present invention includes systems to practice the methods of theinvention. The systems can include hardware to carry out the transfersand provide conditions necessary to practice methods of the invention.The systems can include, e.g., computers, compositions of the invention,controlled heating plates, any of various solid supports, particlesorters, detector systems, fluid handling systems, and/or the like.

In the amplification systems, a nucleic acid of interest in a first bDNAamplification can be detected as the presence of an amplificationoligomer in a second amplification. For example, a two-stageamplification system to detect a nucleic acid of interest can include 1)a first a bDNA amplification system wherein the nucleic acid of interestis the target nucleic acid, an amplification multimer with a pluralityof sequences complementary to an amplification oligomer sequence; and, anon-enzymatic nucleic acid assay capable of detecting the amplificationoligomer sequence. Detection of the amplification oligomer by thenucleic acid assay in such a system indicates the presence of thenucleic acid of interest in the bDNA amplification. In preferredsystems, the amplification oligomer does not comprise a label. Inpreferred systems, nucleic acid assay after the first amplification is abDNA assay in which the amplification oligomer functions as a targetnucleic acid. In alternate embodiments, the nucleic acid assay can beany nucleic acid assay, e.g., a northern blot, a Southern blot,electrophoresis, light absorbance, and/or the like.

In preferred embodiments of the systems, the amplification oligomer is anucleic acid with sequences complimentary to multiple replicatesequences of an amplification multimer in a first amplification, andwith sequences complimentary to capture a label system in a secondamplification. For example, the amplification oligomer can include asequence complimentary to an M-2 sequence of a first amplificationmultimer (functioning in a first amplification), to an L-1 sequence of alabel extender or to an M-1 sequence of a second amplification multimer,and (optionally) to a C-3 sequence of a capture extender or to a C-2sequence of a capture probe of a second amplification capture system.

First and/or second amplifications can take place on the surface of asolid support in systems of the invention. The solid supports can be inthe form of surfaces, beads, particles, microspheres, conduit surfaces,chamber walls, multi-well plates, etc. The solid supports can be formedfrom, glass, silicon, silica, quartz, plastic, polystyrene, nylon, ametal, a ceramic, nitrocellulose, and/or the like. For example, bDNAamplifications or nucleic acid assays can take place on solid supports,such as, e.g., a bead (e.g., microsphere), a bead comprising afluorescent dye, a paramagnetic bead, a multiwell plate, a membrane,and/or the like.

The systems of the invention can function in the multiplexing anddeconvolution of multiple nucleic acid amplifications for the samesample at the same time. The systems can capture and amplify two or moredifferent nucleic acids of interest in a first amplificationaccumulating two or more different amplification oligomers and detectingthe oligomers separately, e.g., at different solid support locations ina second amplification and detection. The system can include two or moredifferent amplification multimers that each accumulate a differentamplification oligomer in association with different nucleic acids ofinterest present in a sample. For example, the system can include afirst amplification multimer that hybridizes directly or indirectly to afirst nucleic acid of interest and has one or more first M-2 sequencesdifferent from second M-2 sequences of a second amplification multimerthat hybridizes to the second of the different nucleic acid sequences ofinterest, so that the two nucleic acids of interest can be separatelyamplified to accumulate different amplification oligomers. A firstamplification oligomer can comprise a sequence complimentary to thefirst M-2 sequence and comprise a sequence complimentary to a first C-3or C-2 sequence, and a second amplification oligomer can comprising asequence complimentary to the second M-2 sequence and comprise asequence complimentary to a second C-3 or C-2 sequence so that thepresence or absence of the first nucleic acid of interest can bedetected based on detectable hybridization of the first amplificationoligomer to the first C-3 or C-2 sequence, and/or the presence orabsence of the second nucleic acid of interest can be detected based ondetectable hybridization of the second amplification oligomer to thesecond C-3 or C-2 sequence. The unique C-2 or C-3 sequence complimentsof the amplification oligomers can allow their direction to be capturedat specific locations on a solid support of a second amplification. Forexample, the first C-3 or C-2 sequences can be bound to solid supportparticles emitting a first identification signal and the second C-3 orC-2 sequences are bound to particles emitting a second identificationsignal different from the first signal, so that the identity of theparticle can be associated with the identity of the nucleic acid ofinterest present in the sample. Alternately, the first C-3 or C-2sequences can be bound to a surface of a first chamber and the secondC-3 or C-2 sequences are bound a surface of a second chamber, so thatsignal ultimately detected from the first chamber indicates the presenceof the first nucleic acid of interest in the sample, and signalultimately from the second chamber indicates the presence of the secondnucleic acid of interest in the sample. Where matrixed amplificationreactions are finally detectable as a signal from an array of beads, aflow cytometer can be used to sort and detect a particle comprising anamplification oligomer; or where the detectable signal is ultimatelyprovided at an array location on a solid support, a charge coupleddevice can be useful for imaging a location of an amplification oligomerin a second amplification.

In another aspect of multiplexed two-stage amplifications, the signalproduced in the second amplification can be deconvoluted bydistinguishing between two or more different signals emanating from thesame solid support location. For example, two or more differentamplification oligomers previously accumulated and melted from a firstamplification solid support can be identified by providing a first labelsystem with a first sequence complimentary to a first label probecomprising a first detectable marker; providing a second label systemcomprising a second sequence complimentary to a second label probecomprising a second detectable marker different from the firstdetectable marker; providing a sample from the first amplification inwhich a first amplification oligomer is capable of hybridizing to acomponent of the first label system, or which sample comprises or issuspected of comprising a second amplification oligomer capable ofhybridizing to a component of the second label system; hybridizing thesample with the first and second label system; and, detecting a signalfrom the first detectable marker or from the second detectable marker.If a signal is detected from the first detectable marker, the presenceof the first amplification oligomer in the sample is indicated. If asignal is detected from the second detectable marker, the presence ofthe second amplification oligomer in the sample is indicated. Inpreferred embodiments of this multiplexed amplification, the firstand/or second label system comprises: a) a label probe comprising asequence complimentary to an amplification multimer having a sequencecomplimentary to a label extender, and the label extender also has asequence complimentary to the first or the second amplificationoligomer, b) a label probe comprising a sequence complimentary to anamplification multimer, which amplification multimer comprises asequence complimentary to the amplification oligomer, and/or c) a labelprobe comprising a sequence complimentary to an amplifier, whichamplifier comprises a sequence complimentary to a preamplifier, whichpreamplifier comprises a sequence complimentary to the amplificationoligomer. In many embodiments, it is preferred that label systemaccomplishes hybridization to the amplification oligomers indirectly,e.g., through label extenders. The first and second label systems canhave the same type of label but with different distinguishable signals,or the first and second label systems can have different types oflabels. For example, different label types can be fluorophores,radionuclides, ligands, enzymes, chromophores, chemiluminescentcompounds, and/or the like. The different distinguishable signals can befluorescent or chemiluminescent emissions at different frequencies,intensities, or combinations of frequencies; radionuclides emittingdifferent particles or with different energies; ligands with differentreporters; enzymes with different substrates or products; chromophoreswith different absorbances, and/or the like. In an aspect of multiplexdeconvolution, the present invention provides identification of two ormore amplification oligomers at substantially the same location bydetecting both a first and second label probe at the location.Optionally, identification is provided for two or more amplificationoligomers at the same time by detecting both the first and seconddetectable marker at substantially the same time; optionally, at thesame solid support location.

In still another aspect of multiplexed two-stage amplifications, thefirst amplification takes place at two or more solid support locationswith different capture systems. The capture systems can uniquelyhybridize with different nucleic acids of interest. In this way,different target nucleic acids of interest from the same sample can becaptured separately then amplified, e.g., using the same set ofamplification multimers and amplification oligomers. After optionallywashing away residual components of the first amplification, a secondamplification can be provided wherein the amplification oligomers at theseparate locations act as the target nucleic acid input to accumulatedetectable label probes at the locations. In one embodiment, the solidsupport locations are on separate beads having identifying signals.After the first hybridization, the beads can be separated to differentpositions for individual second amplifications and detections. In oneembodiment using first captures of target nucleic acids at separate orseparable locations, the nucleic acids of interest are detected by: a)providing a sample comprising or suspected of comprising one or morenucleic acids of interest; b) providing a first solid support whichcomprises first capture extender sequence or first capture probesequence complimentary to a first nucleic acid of interest; c) providinga second solid support which comprises a second capture extendersequence or a second capture probe sequence complimentary to a secondnucleic acid of interest; d) contacting the first and second solidsupports with the sample; e) capturing the first nucleic acid ofinterest on the first solid support and/or capturing the second nucleicacid of interest on the second solid support; f) hybridizing a firstlabeling system directly or indirectly to the first and/or secondnucleic acid of interest; g) hybridizing the first labeling system toone or more amplification oligomers; h) separating the first solidsupport from the second solid support; and i) separately detecting thepreviously hybridized amplification oligomers on each solid support in anucleic acid assay. This technique allows two or more different targetnucleic acids of interest to be separately detected and quantitatedusing unique capture systems but common amplification and detectionsystems.

In two-stage multiplexed amplifications where the first amplificationsolid supports are separate or separable, the detection of the firstamplification can be through a bDNA assay, PCR, LCR, a northern blot, aSouthern blot, electrophoresis, light absorbance and/or the like.Separating steps can be accomplished, e.g., by particle sorting, cellsorting, magnetic sorting, and/or the like.

In another aspect of two-stage multiplexed amplifications with separatefirst amplifications, the amplification oligomers associated through anamplification multimers with the first nucleic acid of interest can bethe same amplification oligomers associated with the second nucleic acidof interest, yet the two nucleic acids can still be separatelyidentified. This result follows from the fact that the chain of identityfrom nucleic acid of interest to label probe signal in this case doesnot depend on the specific capture of the amplification oligomers at aspecific solid support location for the second amplification.

DEFINITIONS

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein havemeanings commonly understood by those of ordinary skill in the art towhich the present invention belongs.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularcompositions, methods, or systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a”, “an” and “the” include plural referents unlessthe content clearly dictates otherwise. Thus, for example, reference to“a component” can include a combination of two or more components;reference to “a nucleic acid” can include mixtures of nucleic acids, andthe like.

Although many methods and systems similar, modified, or equivalent tothose described herein can be used in the practice of the presentinvention without undue experimentation, many preferred materials andmethods are described herein. In describing and claiming the presentinvention, the following terminology will be used in accordance with thedefinitions set out below.

The term “about” as used herein indicates the value of a given quantityvaries by +/−10% of the value, or optionally +/−5% of the value, or insome embodiments, by +/−1% of the value so described.

The term “amplification” in the context of the present inventions,refers to an accumulation of two or more molecules in a system, whichaccumulation is specifically associated with the presence of a nucleicacid of interest (e.g., target nucleic acid of a sample in a firstamplification or amplification oligomer in a second amplification) inthe system (e.g., in solution or at a surface). The accumulation canconsist of enzymatic replication of copies the nucleic acid of interest.Alternately, the amplification can consist of an accumulation of anothermolecule (e.g., binding an amplification product nucleic acid to a solidsupport) dependent on the presence of the nucleic acid of interest. Forexample, in a typical bDNA assay, the presence of a target nucleic acidof interest in the system can be amplified into a large number ofdetectable label probes (or amplification oligomers) bound to a solidsupport in association with the initial presence of the target nucleicacid in a sample. In a typical PCR reaction, the presence of a targetnucleic acid of interest in the system can result in the accumulation ofa large number of replicate copies and complimentary nucleic acidsequences.

The term “polynucleotide” (and the equivalent term “nucleic acid”)encompasses any physical string of monomer units that can becorresponded to a string of nucleotides, including a polymer ofnucleotides (e.g., a typical DNA or RNA polymer), peptide nucleic acids(PNAs), modified oligonucleotides (e.g., oligonucleotides comprisingnucleotides that are not typical to biological RNA or DNA, such as2′-O-methylated oligonucleotides), and the like. The nucleotides of thepolynucleotide can be deoxyribonucleotides, ribonucleotides or polymersof nucleotide analogs, can be natural or non-natural, and can beunsubstituted, unmodified, substituted or modified. The nucleotides canbe linked by phosphodiester bonds, or by phosphorothioate linkages,methylphosphonate linkages, boranophosphate linkages, or the like. Thepolynucleotide can additionally comprise non-nucleotide elements such aslabels, quenchers, blocking groups, or the like. The polynucleotide canbe, e.g., single-stranded or double-stranded.

A “polynucleotide sequence” or “nucleotide sequence” is a polymer ofnucleotides (an oligonucleotide, a DNA, an RNA, a nucleic acid, etc.) ora character string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence (e.g., thecomplementary nucleic acid) can be determined.

Two polynucleotides “hybridize” when they associate to form a stableduplex, e.g., under relevant assay conditions. Nucleic acids hybridizedue to a variety of well characterized physico-chemical forces, such ashydrogen bonding, solvent exclusion, base stacking and the like. Anextensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, part I chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays” (Elsevier, N.Y.).

The “Tm” (melting temperature) of a nucleic acid duplex under specifiedconditions (e.g., relevant assay conditions) is the temperature at whichhalf of the base pairs in a population of the duplex are disassociatedand half are associated. The Tm for a particular duplex can becalculated and/or measured, e.g., by obtaining a thermal denaturationcurve for the duplex (where the Tm is the temperature corresponding tothe midpoint in the observed transition from double-stranded tosingle-stranded form).

The term “complementary” refers to a polynucleotide that forms a stableduplex with its “complement,” e.g., under relevant assay conditions.Typically, two polynucleotide sequences that are complementary to eachother have mismatches (mismatched base pairs) at less than about 20% ofthe bases, at less than about 10% of the bases, preferably at less thanabout 5% of the bases, one mismatch, and more preferably have nomismatches.

A “capture probe” or “CP” is a polynucleotide attached to a solidsupport and comprises a sequence useful to directly or indirectlyspecifically capture a particular nucleic acid of interest. For example,a capture probe can specifically hybridize to a capture extender (and/orcan include a sequence complimentary to a nucleic acid of interest,e.g., to directly and specifically capture the nucleic acid of interest)and that is tightly bound (e.g., covalently or non-covalently, directlyor through a linker, e.g., streptavidin-biotin or the like) to a solidsupport, a spatially addressable solid support, a slide, a particle, amicrosphere, a bead, or the like. The capture probe typically comprisesat least one polynucleotide sequence C-2 that is complementary topolynucleotide sequence C-1 of at least one capture extender (or, insystems designed for direct capture of a nucleic acid of interest, theC2 sequence can be complimentary to a sequence of the nucleic acid ofinterest). The capture probe is preferably single-stranded.

A “capture extender” or “CE” is a polynucleotide (or comprises apolynucleotide) that is capable of hybridizing to a nucleic acid ofinterest and to a capture probe. A capture extender can bind aparticular nucleic acid of interest to a particular solid support,through a capture probe, with high specificity. The capture extendertypically has a first polynucleotide sequence C-1, which iscomplementary to the capture probe, and a second polynucleotide sequenceC-3, which is complementary to a polynucleotide (target) sequence of thenucleic acid of interest. Sequences C-1 and C-3 are typically notcomplementary to each other. The capture extender is preferablysingle-stranded.

A “label extender” or “LE” is a polynucleotide that is capable ofhybridizing to a nucleic acid of interest and to a label probe system. Acapture extender can link a particular nucleic acid of interest tocomponents of a label system. The label extender typically has a firstpolynucleotide sequence L-1, which is complementary to a polynucleotidesequence of the nucleic acid of interest, and a second polynucleotidesequence L-2, which is complementary to a polynucleotide sequence of thelabel probe system (e.g., L-2 can be complementary to an M-1polynucleotide sequence of an amplification multimer, a preamplifier, alabel probe, or the like). The label extender is preferablysingle-stranded.

A “label” is a moiety that facilitates detection of a molecule (e.g., byproviding a detectable signal). Common labels in the context of thepresent invention include fluorescent, luminescent, light-scattering,and/or colorimetric labels. Suitable labels include enzymes andfluorescent moieties, as well as radionuclides, substrates, cofactors,inhibitors, chemiluminescent moieties, magnetic particles, and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Many labels are commercially available and can be used in thecontext of the invention.

A “label probe” or “LP” is a single-stranded polynucleotide thatcomprises a label (or optionally that is configured to bind to a label)that directly or indirectly provides a detectable signal. The labelprobe typically comprises a polynucleotide sequence that iscomplementary to the repeating polynucleotide sequence M-2 of anamplification multimer. Optionally, a label probe can hybridize directlyto a sequence on the nucleic acid of interest (e.g., an amplificationoligomer). However, in most cases, label probes are not designed tohybridize directly to the nucleic acid of interest.

A “label probe system”, in the context of the present inventions,comprises one or more polynucleotides that can hybridize (through alabel extender or not), to associate one or more labels with a nucleicacid of interest. The nucleic acid of interest can be, e.g., a targetnucleic acid of interest in a sample or an amplification oligomer targetof a second amplification of the methods. For example, a label probesystem can comprise a combination of label extenders, amplificationmultimers, preamplifiers, amplifiers and/or label probes. In oneembodiment, the label probe system comprises an amplification multimerwith an M-1 sequence complimentary to a label extender and a pluralityof M-2 sequences complimentary to a label probe sequence. Optionally,the label probe system comprises a preamplifier with a sequencecomplimentary to a label extender L-2 sequence and replicate sequencescomplimentary to an amplifier sequence, which amplifier also hasreplicate sequences complimentary to a label probe sequence. One or morepolynucleotide sequences M-1 of the label probe system are optionallyidentical sequences or different sequences. Optionally, the label probesystem hybridizes directly to a nucleic acid of interest without a labelextender. Alternately, the label probe system is simply a label probewith a sequence complimentary to a nucleic acid of interest (or not).The label probe system can include a plurality of label probes (e.g., aplurality of identical label probes, probes of different types, orprobes of the same type but providing different detectable signals).

A “complex” in the context of amplifications of the invention, refers tothree or more amplification components (e.g., capture probes, captureextenders, label extenders, amplification multimers, amplificationoligomers, or label probes) bound together through hybridization ofcomplimentary sequences.

“Different” polynucleotides have different sequences. For example, apolynucleotide or polynucleotide sequence is different from another ifthey do not have 100% sequence identity, have less that 99% identity,less that 95% identity less than 90% identity or less than 80% sequenceidentity.

Polynucleotides are “captured” when they are bound directly orindirectly (e.g., through hybridization to an extender or as part of acomplex) to a solid support.

Polynucleotides are “indirectly” associated (e.g., hybridized, captured,bound) with a solid support or another identified polynucleotide whenthe association comprises linkage through one or more otherpolynucleotide, such as, e.g., a capture extender or label extender.

An “amplification multimer” is a polynucleotide comprising a sequencedirectly complimentary to a nucleic acid of interest (or indirectlyspecifically hybridizable to the nucleic acid of interest, e.g., througha label extender) and comprising a plurality of substantially identicalpolynucleotide sequences complimentary to label probes (or toamplification oligomers, in the case of a first amplification of atwo-stage amplification). The amplification multimer has a structuredesigned to function by specifically associating multiple label probesor amplification oligomers with a nucleic acid of interest. For example,amplification multimers can have an M-1 polynucleotide sequencecomplimentary to a nucleic acid of interest and a plurality of M-2sequences complimentary to label probes, e.g., to specifically bindmultiple labels to the nucleic acid of interest (e.g., an amplificationoligomer). Alternately, amplification multimers can have an M-1polynucleotide sequence complimentary to a nucleic acid of interest(e.g., a test sample nucleic acid) and a plurality of sequences M-2complimentary to amplification oligomers, e.g., to specificallyaccumulate multiple amplification oligomers in association with thenucleic acid of interest. In many embodiments of two-stage non-enzymaticamplifications of the invention, a first amplification multimer of afirst amplification stage comprises an M-1 sequence complimentary to asample target nucleic acid of interest (or indirectly hybridizable tothe sample target nucleic acid of interest through a label extender) andmultiple replicate M-2 sequences complimentary to an amplificationoligomer; and, a second amplification multimer of a second stageamplification comprising an M-1 sequence complimentary to theamplification oligomer (or indirectly hybridizable to the amplificationoligomer through a label extender) and multiple replicate M-2 sequencescomplimentary to a label probe. The M-1 sequence of the amplification isnot necessarily attached to the replicate M-2 sequences solely throughcovalent bonds (i.e., M-2 sequences of an amplification multimer can beassociated with M-1 sequences through non-covalent interactions, suchas, e.g., polynucleotide hybridizations, affinity interactions, and/orthe like). For example, an amplification multimer can comprise apreamplifier complimentary to a label extender (or target nucleic acid)and an amplifier complimentary to the preamplifier and multiple labelprobes (or amplification oligomers). The amplification multimer can be,e.g., a linear or a branched nucleic acid. As noted for allpolynucleotides, the amplification multimer can include modifiednucleotides and/or nonstandard internucleotide linkages as well asstandard deoxyribonucleotides, ribonucleotides, and/or phosphodiesterbonds. Suitable amplification multimers are described, for example, inU.S. Pat. No. 5,635,352, U.S. Pat. No. 5,124,246, U.S. Pat. No.5,710,264, and U.S. Pat. No. 5,849,481.

An amplification oligomer can be designed to have sequences allowing itto function both as the amplification product of a first branched DNAamplification and as the input target of a second nucleic acidamplification. As used herein with regard to a two-stage amplificationmethod, the term “amplification oligomer” refers to a polynucleotidecomprising a sequence complimentary to the repeat sequencesamplification multimers employ to accumulate polynucleotides (e.g.,amplification multimer repeat sequences analogous to those that capturelabel probes in classic bDNA assays, e.g., M-2 sequences) of a firstbDNA amplification and also comprising sequences complimentary to 1) alabel system component (e.g., label extenders (e.g., at L-1) oramplification multimers (e.g., at M-1)) of a second bDNA amplification,or 2) one or more primer oligomers of an enzymatic amplificationtechnique. An amplification oligomer can be designed with sequencesallowing it to function as both the amplification product of a firstbDNA amplification and as the target nucleic acid of interest in asecond bDNA amplification. For example, in the context of non-enzymatictwo-stage amplifications of the invention an “amplification oligomer” isdesigned to function in a stringent hybridization with at least twosequence replicates of the amplification multimer of the firstamplification and also designed to function in a stringent hybridizationto capture the label system complex of the second amplification.

A “preamplifier” is a nucleic acid that serves as an intermediatebetween one or more label extenders and amplifiers. Typically, thepreamplifier is capable of hybridizing simultaneously to at least twolabel extenders and to a plurality of amplifiers.

Branched DNA technology used in the methods can include amplificationmethods that employ label systems that hybridize directly or indirectlyto a target nucleic acid and associate multiple copies of anothernucleic acid (e.g., label probes or amplification oligomers) to thetarget nucleic acid. It is worth noting that the amplification multimeris typically, but not necessarily, a branched-chain nucleic acid; forexample, the amplification multimer can be a branched, forked, orcomb-like nucleic acid or a linear nucleic acid, or a complex thereof.Typically branched DNA of the present invention includes a nucleic acidhaving a “trunk” structure covalently attached to multiple nucleic acidbranches having multiple sequences complimentary to, e.g., label probes.For example, see amplification multimers. Alternately, the branches ofthe branched DNA label systems can be attached to a “trunk” by affinityor hybridization systems to ultimately associate the multiple sequencesto the target nucleic acid. For example, see labeling systems, describedherein, comprising label extenders complimentary to preamplifierscomplimentary to amplifiers complimentary to label probes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of an exemplary old art bDNA assaysystem.

FIGS. 2A and 2B show schematic diagrams depicting a two-stageamplification. In the first amplification of FIG. 2A, target nucleicacid of interest is amplified in a bDNA amplification to accumulate amultitude of amplification oligomers. In the second amplification ofFIG. 2B, one of the amplification oligomers from the first amplificationstage is amplified in a bDNA amplification accumulating a multitude oflabel probes.

FIGS. 3A and 3B are schematic diagrams showing an exemplary two-stageamplification employing cooperative hybridization and multipleamplification multimers for each captured target nucleic acid ofinterest.

FIG. 4 is a schematic diagram of exemplary two-stage amplificationsystem hardware components.

FIGS. 5 A through 5G, present schematic diagrams of a multiplexedtwo-stage amplification using a planar solid support in the firstamplification and microsphere solid supports in the secondamplification.

FIG. 6 is a schematic diagram showing how signals detected from amultiplexed two-stage amplifying can be deconvoluted to unambiguouslyassociate the signals with the presence of a particular nucleic acid ina sample.

FIG. 7 is a schematic diagram showing an exemplary embodiment of atwo-step multiplexed amplification. A variety of amplification oligomerswith different zip codes (e.g., compliments to second amplification C-3sequences) are enriched in a first amplification wherein eachamplification multimer has 15 replicate sequences complimentary toaccumulate amplification oligomer sequences.

FIG. 8 is a schematic diagram showing a second amplification stepwherein the variety of amplification oligomers accumulated in the firstamplification (e.g., of FIG. 7) are each amplified 400-fold onamplification multimers of specific beads in a second amplification.

DETAILED DESCRIPTION

The present invention provides methods and systems using amplificationoligomers in a two-stage amplification scheme that can detect thepresence of a nucleic acid of interest with improved sensitivity andconsistency in a complex sample. Amplification oligomers are nucleicacids comprising, e.g., sequences designed to be accumulated at a solidsupport by hybridization to repetitive complimentary sequences in afirst non-enzymatic amplification and also comprising sequences actingas the target sequence for a second amplification system or nucleic acidassay. The accumulation of the amplification oligomer in the firstamplification is dependent on the presence of a target nucleic acid ofinterest in a sample so that a detection signal from the secondamplification or assay correlates to the presence of the target nucleicacid in the sample. Two-stage amplifications of the invention candramatically enhance the sensitivity for target nucleic acid detection.Information designed into the amplification oligomers can allowmultiplexed assays that provide sensitive detection and quantitation oftwo or more target nucleic acids from the same sample at once.

Methods and systems using amplification oligomers can enable separateidentification and quantitation of two or more nucleic acids of interestfrom the same sample at the same time. For example, two differentamplification oligomers, representing two different target nucleic acidsof interest, can be enriched in a first amplification. Unique “zip code”sequences in each different amplification oligomer (e.g., sequencescomplimentary to predesignated capture probes) can allow capture andamplification of the different amplification oligomers at differentsolid support locations. Thus, the different amplification oligomers,representing target nucleic acids from the same sample, can beseparately amplified, identified and quantitated.

Two-Stage Amplifications Detecting Nucleic Acids of Interest

Two-stage amplification methods of the invention generally include anon-enzymatic amplification seeded by a target nucleic acid of interestfollowed by another non-enzymatic amplification seeded by theamplification product of the first amplification. Typically, theamplification product of the first amplification is an amplificationoligomer, e.g., having sequences useful in targeting the oligomer to anintended solid support and having sequences capable of specificallyinteracting with a second amplification system. Optionally, the secondamplification can be an enzymatic amplification known in the art, e.g.,to replicate target nucleic acids. The present invention is not limitedto these preferred embodiments and can be practiced in a variety ofways.

In many embodiments of the methods at least the first amplificationstage relies on branched DNA (bDNA) technology. In a typical case of abDNA amplification, shown in FIG. 1, e.g., a target nucleic acid 114 iscaptured by a capture extender 111 associated with a solid support 101,the target is decorated at one or more locations with label extenders121 typically associated with branched DNA molecules 141 capable ofbinding multitude label probes 142 to generate a large signal associatedwith the initial capture of a small amount of target.

In the present invention, amplification oligomers are the product of afirst amplification dependent on the presence of the target nucleic acidinstead of a label probe product. The large amount of amplificationoligomers accumulated in association with the target nucleic acid in thefirst amplification stage can then act as the target nucleic acid in asecond amplification. For example, as shown in FIG. 2A, a target nucleicacid 214 is captured from a sample onto a solid support 201. Anamplification multimer 241 can then be captured on the solid support byhybridization to complimentary target nucleic acid sequences. Theamplification multimer can, in turn, capture a large number ofamplification oligomers 242. With the amplification oligomer productcaptured at the first solid support, the hybridization solution andunbound components of the first amplification system can be washed awaywith wash solutions at temperatures of appropriate stringency. Then, theamplification oligomers can be melted from the first solid support andtransferred to a second amplification system, e.g., associated with asecond solid support.

As shown in FIG. 2B, the amplification oligomer can act as the targetnucleic acid in the second amplification to accumulate a large number oflabeled probes. The signal from these label probes provides a highlysensitive determination of target nucleic acid in the initial sample.For example, Amplification oligomer 242 transferred to the secondamplification system can be captured by hybridization to the secondsolid support 261 through sequences complimentary to capture probes 264on the solid support. Amplification multimers 265 can be captured byhybridization to sequences complimentary to the captured amplificationoligomers and label probes 262 can be captured in large numbers atcomplimentary sequences of the amplification multimer.

Through the course of the two amplifications, a single target nucleicacid of interest captured from the sample on the first solid support canultimately lead to the specific capture of many-fold more label probesin the second amplification. For example, as shown in FIG. 3A, in afirst amplification, a single target nucleic acid molecule of interest300 can capture, e.g., six amplification multimers 301. Eachamplification multimer can capture, e.g., 14 amplification oligomers302, for an initial 84-fold amplification of the target to amplificationoligomers. As shown in FIG. 3B, the amplification oligomers 302 of thefirst amplification can be transferred, e.g., to be captured on a secondsolid support 304 to act as a nucleic acid of interest in a secondamplification. Here, one amplification oligomer 302, of the 84accumulated in the first amplification, can capture, e.g., twoamplification multimers 305, which can accumulate 45 label probes 306each, for a 90-fold amplification of each amplification oligomers tolabel probe amplification product. Over all, through two amplificationsin this example, there has been a 756-fold (84×90) molecularamplification from the sample target nucleic acid to the label probes.One skilled in the art will appreciate that further amplification can beobtained, e.g., by increasing the number of amplification multimerscaptured in the first and/or second amplification, increasing the numberof replicate sites complimentary to amplification oligomers on the firstamplification multimers, and/or increasing the number of replicate sitescomplimentary to label probes on the second amplification multimers.Furthermore, one skilled in the art will appreciate that furtheramplification can be obtained by inserting one or more additionalamplifications between the first and second amplifications wherein theaccumulated amplification oligomers of the first amplification functionas target for the inserted amplification and the product of the insertedamplification is an amplification oligomer functioning as target for asubsequent amplification.

In the preceding example, label probe system comprises the secondamplification multimer and the label probes (with optional labelextenders). In other embodiments, the label probe system could comprisea preamplifier system, e.g., as described in U.S. Pat. No. 5,635,352 andU.S. Pat. No. 5,681,697, which can typically provide even largeramplification factors at each amplification. In yet another example, thelabel extenders hybridize directly to the label probes and noamplification multimer or preamplifier is used, so the signal from asingle target nucleic acid molecule is only amplified by the number ofdistinct label extenders that hybridize to that nucleic acid.

Basic bDNA assays have been well described and used, e.g., to detecttaggants, to analyze forensic samples, to detect and quantify mRNAtranscripts in cell lines, to determine viral loads, and the like. ThebDNA assay provides reliable direct quantification of nucleic acidmolecules at physiological levels. Several advantages of the technologydistinguish it from other DNA/RNA amplification technologies, includinglinear amplification, good sensitivity and dynamic range, greatprecision and accuracy, simple sample preparation procedure, and reducedsample-to-sample variation. For additional details on bDNA assays, see,e.g., U.S. Pat. No. 4,868,105 to Urdea et al. entitled “Solution phasenucleic acid sandwich assay”; U.S. Pat. No. 5,635,352 to Urdea et al.entitled “Solution phase nucleic acid sandwich assays having reducedbackground noise”; U.S. Pat. No. 5,681,697 to Urdea et al. entitled“Solution phase nucleic acid sandwich assays having reduced backgroundnoise and kits therefore”; U.S. Pat. No. 5,124,246 to Urdea et al.entitled “Nucleic acid multimers and amplified nucleic acidhybridization assays using same”; U.S. Pat. No. 5,624,802 to Urdea etal. entitled “Nucleic acid multimers and amplified nucleic acidhybridization assays using same”; U.S. Pat. No. 5,849,481 to Urdea etal. entitled “Nucleic acid hybridization assays employing largecomb-type branched polynucleotides”; U.S. Pat. No. 5,710,264 to Urdea etal. entitled “Large comb type branched polynucleotides”; U.S. Pat. No.5,594,118 to Urdea and Horn entitled “Modified N-4 nucleotides for usein amplified nucleic acid hybridization assays”; U.S. Pat. No. 5,093,232to Urdea and Horn entitled “Nucleic acid probes”; U.S. Pat. No.4,910,300 to Urdea and Horn entitled “Method for making nucleic acidprobes”; U.S. Pat. No. 5,359,100; U.S. Pat. No. 5,571,670; U.S. Pat. No.5,614,362; U.S. Pat. No. 6,235,465; U.S. Pat. No. 5,712,383; U.S. Pat.No. 5,747,244; U.S. Pat. No. 6,232,462; U.S. Pat. No. 5,681,702; U.S.Pat. No. 5,780,610; U.S. Pat. No. 5,780,227 to Sheridan et al. entitled“Oligonucleotide probe conjugated to a purified hydrophilic alkalinephosphatase and uses thereof”; U.S. patent application Publication No.US2002172950 by Kenny et al. entitled “Highly sensitive gene detectionand localization using in situ branched-DNA hybridization”; Wang et al.(1997) “Regulation of insulin preRNA splicing by glucose” Proc Nat AcadSci USA 94:4360-4365; Collins et al. (1998) “Branched DNA (bDNA)technology for direct quantification of nucleic acids: Design andperformance” in Gene Quantification, F Ferre, ed.; and Wilber and Urdea(1998) “Quantification of HCV RNA in clinical specimens by branched DNA(bDNA) technology” Methods in Molecular Medicine: Hepatitis C 19:71-78.In addition, reagents for performing basic bDNA assays (e.g.,QuantiGene™ kits, amplification multimers, alkaline phosphatase labeledlabel probes, chemilumigenic substrate, capture probes immobilized on asolid support, and the like) are commercially available, e.g., fromPanomics, Inc. (on the world wide web at www.panomics.com), and can beadapted for the practice of the present invention. Software fordesigning probe sets for a given nucleic acid target (i.e., fordesigning the regions of the capture extenders, label extenders, andoptional blocking probes that are complementary to the target) is alsocommercially available (e.g., ProbeDesigner™ from Panomics, Inc.); seealso Bushnell et al. (1999) “ProbeDesigner: for the design of probe setsfor branched DNA (bDNA) signal amplification assays Bioinformatics15:348-55.

In some embodiments of the methods, the second amplification can be anenzymatic amplification. For example, the amplification oligomer productof the first amplification can be input as a target nucleic acid in aligase chain reaction (LCR) or polymerase chain reaction (PCR) for thesecond amplification and detection. In a preferred embodiment,accumulated amplification oligomers from a first branched chainamplification can be melted from the amplification multimers andtransferred to a PCR reaction chamber. In PCR amplifications, theamplification oligomer can function as the PCR template region to beamplified. Two or more different primers are included in the PCRreaction solution to determine the beginning and end of the region to beamplified. Taq polymerase can then incorporate and nucleotidetriphosphates to copy the region between the primers by primerextensions. The PCR process is carried out in a thermal cycler thatheats and cools the reaction chamber according to a programmabletemperature profile cycle required for target melting, primerhybridization to target, primer extension, and replicate melting. Thecycle typically continues up to about 35 cycles for an amplificationranging from about 10³ to about 10⁶-fold. The amplification product canbe detected, e.g., by incorporation or labeled nucleotides into theamplification products during the primer extensions, or by otherconventional means, such as, e.g., northern blotting, a Southernblotting, electrophoresis, 280 nm absorbance, and the like.

Samples Containing Target Nucleic Acids of Interest

The two-stage amplification methods of the invention can amplify, andultimately detect nucleic acids of interest with high sensitivity fromcomplex sample materials. The nucleic acids can be any type of interest,such as, e.g., DNA, cDNA, rRNA, mRNA, miRNA, etc.

The methods can be used to detect the presence of the nucleic acids ofinterest in essentially any type of sample. For example, the sample canbe derived from an animal, a human, a plant, a cultured cell, a virus, abacterium, a pathogen, and/or a microorganism. The sample optionallyincludes a cell lysate, an intercellular fluid, a bodily fluid(including, but not limited to, blood, serum, saliva, urine, sputum, orspinal fluid), and/or a conditioned culture medium, and is optionallyderived from a tissue (e.g., a tissue homogenate), a biopsy, and/or atumor. As just a few examples, the nucleic acids of interest can bederived from one or more of an animal, a human, a plant, a culturedcell, a microorganism, a virus, a bacterium, or a pathogen. The use of abDNA type amplification can have a great advantage over many enzymaticamplifications for such complex and impure samples becausehybridizations are less sensitive to interference than typicalamplification enzymes. With the bulk of the complex sample washed awayafter the first amplification, the first amplification product can befar more compatible with second amplification systems, includingenzymatic amplification systems.

The methods can amplify and detect nucleic acids of interest known to bein a sample (e.g., to provide quantitative results) and/or the methodscan detect the presence of a nucleic acid of interest suspected of beingpresent in a sample. To test for the presence of a nucleic acidsuspected of being in a sample, the first amplification stage, e.g.,would provide capture extenders and label extenders complimentary tothat sample nucleic acid of interest. If the nucleic acid of interest ispresent in the sample, the first amplification would be productive andprovide amplification oligomers in amounts adequate to provide apositive signal in the second amplification. If the nucleic acid ofinterest is not present in the sample, or is present in amounts belowthe sensitivity of the two-stage amplification system, inadequateamounts of amplification oligomer wound be accumulated in the firstamplification and no positive signal above background levels would bedetected for the second amplification. Thus, the two-stage amplificationcan be configured to detect a nucleic acid of interest suspected ofbeing present in a sample, and can confirm whether or not the nucleicacid is actually present with a high sensitivity.

The methods can be used to quantitatively detect nucleic acids ofinterest in samples, e.g., for gene expression analysis. Accordingly, inone class of embodiments, the one or more nucleic acids of interestcomprise one or more mRNAs. A standard curve of, e.g., signal outputfrom the second amplification versus known amounts of mRNA input to thefirst amplification can be prepared. The output signal associated withthe one or more mRNAs can be compared to the standard curve to determinethe amount of the mRNAs in an unknown sample, as is known in the art.

Capturing Nucleic Acids

In many methods of the invention, nucleic acids of interest are capturedon a solid support, e.g., in a step of the first and/or secondamplification. The nucleic acids are typically in a purified or crudesolution, allowing them to kinetically interact with groups associatedwith a solid support surface. The nucleic acids of interest can becaptured directly or indirectly, specifically or non-specifically. Inpreferred embodiments, the nucleic acids are captured at the solidsupport of the first amplification indirectly (e.g., through captureextenders) with a high degree of specificity (e.g., under stringenthybridization conditions).

Nucleic acids of interest can be captured on a solid support directlyand non-specifically. For example, the nucleic acids can be capturedthrough chemical reactions or non-specific chemical interactions betweenthe solid support and the nucleic acid. The solid support can includereactive groups that form covalent bonds to bases, or preferably thesugar-phosphate chain of the nucleic acid. The solid support can includechemical groups that interact with the nucleic acids throughnon-covalent forces, such as, e.g., ionic interactions, hydrophobicinteractions, chelation, Van der Waals forces, polar interactions,and/or the like. A typical solid support for direct non-specific captureof nucleic acids can be, e.g., nitrocellulose or nylon membranesotherwise used in dot-blot or Southern blot analyses. Direct,non-specific capture is most appropriate, e.g., when the sample isrelatively pure and/or the nucleic acid of interest is expected to be apredominant nucleic acid in the sample. In methods using direct,non-specific capture, it can be important to block the solid support,e.g., with sheared salmon sperm DNA and/or the like, to avoid generationof non-specific background signals.

In other embodiments, the nucleic acids of interest can be captureddirectly and specifically on a solid support. For example, the solidsupport can comprise a nucleic acid (e.g., capture probe) on thesurface, which has sequences complimentary to the nucleic acid ofinterest. Exposure of the nucleic acid of interest, in a sample solutionadjusted to appropriate stringency, to a capture probe on the solidsupport can result in capture by specific hybridization directly withthe solid support. Such an arrangement is capable of capturing thenucleic acid of interest from a complex sample, such as a lysate, evenif the nucleic acid of interest represents only a small minority of thenucleic acids in the sample. Optionally, nucleic acids, (e.g.,comprising haptens or protein binding sites) can be capturedspecifically and directly by nucleic acid binding proteins orantibodies.

In still other embodiments, the nucleic acid of interest can be capturedindirectly and specifically. In a preferred embodiment, a single type ofsolid support has the flexibility to optionally capture variousalternate nucleic acids of interest. Such a solid support can have a“universal” capture probe that can hybridize to any number of captureextender oligonucleotides having a sequence complimentary to the captureprobe sequence and designed to be specifically to a particular nucleicacid of interest. In this way, the nucleic acid of interest can becaptured indirectly, through the capture extender, but specificallythrough hybridization to the complimentary sequence of the captureextender. The solid support is universal and the capture extenderprovides specificity to indirectly capture the nucleic acid.

Hybridizing Nucleic Acids

Hybridizing is an important aspect of the first and/or secondamplifications in the methods of the invention. Hybridizations betweencomplimentary sequences of amplification reaction components canprovide, e.g., the direct or indirect capture of target nucleic acids;the binding interactions of amplification multimers, amplificationoligomers and/or label probes; retention of desired components whileundesired materials are removed in stringent wash steps; accumulation ofsubstrates for enzyme amplifications; and/or the like.

Nucleic acids “hybridize” when they specifically associate in solutionappropriate conditions. Nucleic acids hybridize due to a variety of wellcharacterized physico-chemical forces, such as hydrogen bonding, solventexclusion, base stacking and the like. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes, part I, chapter 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays,” (Elsevier,N.Y.), as well as Ausubel et al., eds., Current Protocols, a jointventure between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc., (supplemented through 2003). Hames and Higgins (1995) GeneProbes 1, IRL Press at Oxford University Press, Oxford, England (Hamesand Higgins 1) and Hames and Higgins (1995) Gene Probes 2, IRL Press atOxford University Press, Oxford, England (Hames and Higgins 2) providedetails on the synthesis, labeling, detection and quantification of DNAand RNA, including oligonucleotides.

Methods of the invention can be optimized for hybridization and washingstringency through empirical studies, or through calculations ofpreferred conditions. Stringent hybridization conditions are typicallyat a temperature near the melting temperature (T_(m)) of thecomplimentary sequences involved. For example, in the context of thepresent invention, stringent conditions for a given solution are 10° C.or less below the T_(m), 5° C. or less below the T_(m), 3° C. or lessbelow the T_(m), 1° C. below the T_(m), or at about the T_(m) of thesubject hybridized compliments. The T_(m) of a DNA-DNA duplex can beestimated using the following equation:

T _(m)(° C.)=81.5° C.+16.6(log₁₀ M)+0.41(%G+C)−0.72(%f)−500/n,

where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. See, Rapley and Walker, supra. The T_(m)of an RNA-DNA duplex can be estimated as follows:

T _(m)(° C.)=79.8° C.+18.5(log₁₀M)+0.58(%G+C)−11.8(%G+C)²−0.56(%f)−820/n,

where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. Id. Equations 1 and 2 are typicallyaccurate only for hybrid duplexes longer than about 100-200 nucleotides.Id. The Tm of nucleic acid sequences shorter than 50 nucleotides can becalculated as follows:

T _(m)(° C.)=4(G+C)+2(A+T),

where A (adenine), C, T (thymine), and G are the numbers of thecorresponding nucleotides.

In certain embodiments of the invention all of the hybridizations of theamplification complex are designed to have melting temperatures withinabout 5° C. of each other. In other cases, all the melting temperaturesare designed to be within about 3^(∞) or about 1° C. of each other. Inother embodiments, the melting temperatures of hybridizations in thecomplexes are designed to have higher melting temperatures forhybridizations initiated early in am amplification and lower meltingtemperatures for hybridizations initiated later in the amplificationprocess (e.g., with stringent washes between the hybridizations).

Blocking probes can optionally be hybridized to the nucleic acids ofinterest, e.g., in bDNA amplifications to reduce background in theassay. For a given nucleic acid of interest, the corresponding labelextenders, optional capture extenders, and optional blocking probes arepreferably complementary to physically distinct, non-overlappingsequences in the nucleic acid of interest, which are preferably, but notnecessarily, contiguous. It can be desirable to include blocking probesin bDNA amplifications, which are complimentary to all target nucleicacid sequences not hybridized to other components of the amplificationsystem. The T_(m)s of the capture extender-nucleic acid, labelextender-nucleic acid, and blocking probe-nucleic acid complexes arepreferably greater than the temperature at which the capture extenders,label extenders, and/or blocking probes are hybridized to the nucleicacid, e.g., by 5° C. or 10° C. or preferably by 15° C. or more, suchthat these complexes are stable at that temperature. Potential CE and LEsequences (e.g., potential sequences C-3 and L-1) are optionallyexamined for possible interactions with non-corresponding nucleic acidsof interest, LEs or CEs, the preamplifier, the amplification multimer,the label probe, and/or any relevant genomic sequences, for example;sequences expected to cross-hybridize with undesired nucleic acids aretypically not selected for use in the CEs or LEs. See, e.g., Player etal. (2001) “Single-copy gene detection using branched DNA (bDNA) in situhybridization”, J. Histochem. Cytochem. 49:603-611 and U.S. patentapplication 60/680,976. Examination can be, e.g., visual (e.g., visualexamination for complementarity), computational (e.g., computation andcomparison of binding free energies), and/or experimental (e.g.,cross-hybridization experiments). Capture probe sequences are preferablysimilarly examined, to ensure that the polynucleotide sequence C-1complementary to a particular capture probe's sequence C-2 is notexpected to cross-hybridize with any of the other capture probes thatare to be associated with other subsets of particles or selectedpositions on the support.

Hybridized nucleic acid duplexes align antiparallel. In figures, whereone nucleic acid strand is displayed in one orientation (e.g., 5′ to 3′left to right), the complimentary strand will be in the oppositeorientation (e.g., 3′ to 5′). In complexes of the invention, componentsin serial association are designed to alternate between theseorientations. For example, where a capture probe is attached to a solidsupport from a 3′ end with the 5′ end extending away from the solidsupport, complimentary capture extenders are typically designed toprovide the complimentary sequence on the 5′ end, thus leaving thecapture extender 3′ end free of the solid support surface to capture atarget nucleic acid. Similarly, if a label extender is designed with acomplimentary sequence at the 5′ end intended to hybridize with acaptured nucleic acid of interest (leaving the 3′ LE end free), anamplification multimer compliment strand will typically be designed witha 3′ end sequence free to specifically hybridize to the free 3′ end ofthe label extender.

The various hybridization and capture steps can be performedsimultaneously or sequentially, in any convenient order. For example, inembodiments in which capture extenders are employed, each nucleic acidof interest can be hybridized simultaneously with its correspondinglabel extenders and its corresponding capture extenders in solution, andthen the capture extenders can be hybridized with capture probesassociated with the solid support. Materials not captured on the supportare preferably removed, e.g., by washing the support, and then the labelprobe system is hybridized to the label extenders.

Washing Solid Supports

At any of various steps, materials not captured on the solid support areoptionally separated from the support by washing. For example, after thecapture extenders, nucleic acids, label extenders, blocking probes, andsupport-bound capture probes are hybridized, the support is optionallywashed to remove unbound nucleic acids and probes; after the labelextenders and amplification multimer are hybridized, the support isoptionally washed to remove unbound amplification multimer; and/or afterthe label probes are hybridized to the amplification multimer, thesupport is optionally washed to remove unbound label probe prior todetection of the label. The support is optionally washed at the end ofthe first amplification to remove unbound amplification oligomers.

After hybridization steps in the methods, unhybridized nucleic acidmaterial can be removed by a series of washes, the stringency of whichcan be adjusted depending upon the desired results. Low stringencywashing conditions (e.g., using higher salt and lower temperature) canincrease sensitivity, but can produce nonspecific hybridization signalsand high background signals. Higher stringency conditions (e.g., usinglower salt and a higher temperature that is closer to the hybridizationtemperature) lowers the background signal, typically with only thespecific signal remaining. See, Rapley, R. and Walker, J. M. eds.,Molecular Biomethods Handbook (Humana Press, Inc. 1998), which isincorporated herein by reference in its entirety for all purposes.“Stringent hybridization wash conditions” in the context of theamplification methods, are sequence dependent. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993), supra, and inHames and Higgins 1 and Hames and Higgins 2, supra.

Transferring Amplification Oligomers to a Second Amplification

At the end of the first amplification, the amplification oligomersaccumulated on the first solid support can be melted back into asolution and, optionally, transferred physically to a new location forthe second amplification or nucleic acid assay.

At the end of a typical first amplification, a capture probe on a solidsupport is hybridized to a capture extender, which is hybridized to thetarget nucleic acid, which is hybridized to one or more label extenders,which are hybridized to amplifier multimers, which are hybridized to alarge number of amplification oligomers. See, e.g., FIG. 3A. Any excessamplification components or sample materials are washed away from thesolution surrounding the bound materials on the solid support. Ideally,melting conditions can be provided that substantially melt theamplification oligomers from the amplification multimers while the othernoted hybridizations do not melt. This could be accomplished byproviding a lower T_(m) for the amplification oligomer hybridizationthan for the other hybridizations of the first amplification. With theamplification oligomer melting at a lower temperature from theamplification oligomer, the amplification oligomer could be melted intosolution at a high purity. Highly pure amplification oligomer can undersome circumstances have advantages of lower background and highersensitivity in the second amplification. Alternately, the meltingsolution and temperature can melt all hybridizations of the firstamplification; any background issues resolvable by intelligent choice ofsecond amplification components and component concentrations. Forexample, melting temperatures of system components can be manipulated byselection of complimentary sequence length, sequence (e.g., GC %), useof unnatural base analogs, employment of locked nucleic acids (LNA),etc.

Solutions for melting amplification oligomers from the firstamplification can provide conditions (e.g., low salt and hightemperature) that can melt hybridizations of the amplification oligomer.Optionally, the melting solution can provide conditions, e.g., buffers,salts and components, appropriate to the second amplification reaction.For example, where the second amplification is a bDNA amplification, themelting solution can include the capture extenders, label extenders,amplification multimers and buffer solutions of the second amplificationsystem. In another example, where the second amplification is a PCRreaction, the melting solution can include, e.g., taq polymerase, PCRprimers, nucleotide triphosphates, and/or other components of the PCRreaction.

The solution of amplification oligomer melted from the solid support ofthe first amplification can be transferred by any appropriate meansknown in the art. For example, the solution of unbound amplificationoligomers can be aspirated with pipettes and manually or roboticallytransferred to the surface or chamber of the second amplification. Thesolution of amplification oligomers can be pumped through a conduit offrom the first amplification to the location of the secondamplification.

In certain embodiments, the amplification oligomer accumulated in thefirst amplification does not have to be transferred to a new location(e.g., chamber of surface) in order to carry out the secondamplification. The second amplification can be carried out, for example,by adding components of a second bDNA assay or an enzymaticamplification to the first amplification product, e.g., in the presenceof the first solid support.

For example, after the excess components of the first amplification havebeen washed away, the hybridized components can be melted into thereaction solution of the second amplification containing, e.g., anexcess of second capture extenders, second label extenders secondamplification multimers and label probes. The capture extenders can havesequences complimentary to the same capture probes of the firstamplification but instead of the sequence complimentary to the targetnucleic acid of interest from the sample, the second capture extendercan have a sequence complimentary to sequences of the amplificationoligomer. Alternately, the second capture extenders can have sequencescomplimentary to the amplification oligomers and to a second set ofcapture probes on the first solid support. The second label extenderscan have sequences complimentary to sequences on the amplificationoligomers and to the second amplification multimer, which has manysequences complimentary to label probes. The second amplification can bedriven with a large excess of second amplification components. Judicioususe of blocker probes can minimize interactions of the amplificationoligomers with residual components of the first amplification.

In another example, a second amplification can take place withouttransfer of the accumulated and melted amplification oligomers by addingcomponents a polymerase chain reaction to the amplification oligomers.For example, the solid support of the first amplification can be in anEppendorf tube that fits in a heat block of a thermocycler used forrunning PCR reactions. After the washing step to remove inboundcomponents of the first amplification, the accumulated amplificationoligomer can be melted into a solution containing all components of aPCR reaction. For example, the melting solution can include a PCR primerpair—a first primer complimentary to the amplification oligomer and asecond primer having, e.g., the same sequence as a portion of theamplification oligomer at some point 5′ along the oligomer from thecompliment of the first primer. A polymerase chain reaction using theseprimers would replicate multiple copies of both strands between andincluding the primers.

Detecting Amplification Oligomers

Amplification oligomers accumulated by binding to the first solidsupport in the presence of the target nucleic acid of interest can bedetected by various means to confirm the presence of the target in thesample. In preferred embodiments of the methods, the amplificationoligomer is detected in or after a second non-enzymatic or enzymaticamplification.

In many embodiments of the invention, the amplification oligomers can bedetected without further enzymatic or non-enzymatic amplification steps.For example, the amplification oligomers can be detected by 280 nmabsorbance, fluorometry in the presence of ethidium bromide,polyacrylamide gel electrophoresis, Southern blotting, northernblotting, and the like.

In preferred embodiments, the amplification oligomer is detectedindirectly in a nucleic acid assay. For example, the presence of theamplification oligomer can be confirmed by amplification of theamplification oligomer in a second amplification providing anothernucleic acid as the detectable amplification product. The amplificationoligomer amplification product of the first amplification can bedetected (and optionally quantitated) in a second amplification, suchas, e.g., a PCR amplification, a TaqMan assay, a ligase chain reaction,a branched DNA assay providing a labeled probe amplification product,and the like. A second bDNA amplification providing anotheramplification oligomer can be run before detection of the secondamplification oligomer directly or by an enzymatic or non-enzymaticamplification assay.

In preferred embodiments of the methods, the amplification oligomer doesnot include a label and is not detected by direct detection of a labelon the oligomer (e.g., the amplification oligomer is not a label probe).In preferred methods of the invention, the amplification oligomer is notdetected indirectly as the product of an enzymatically catalyzedreaction (e.g., the presence of amplification oligomer is not detectedthrough the detection of an enzymatic replication product (e.g., PCRproduct) of the amplification oligomer.

In a more preferred embodiment of detecting the amplification oligomerproduct of a first amplification, the amplification oligomer is detectedin a second branched DNA assay. For example, the first solid supportbinding the amplification oligomer is washed and then melted into amelting solution of appropriate stringency for a second amplification.The melting solution can include components of the branched DNA secondamplification, such as, e.g., capture extenders, blocking probes, and alabel system (e.g., label extenders, preamplifiers, amplifiers, and/orlabel probes). It is preferred that capture extenders and labelextenders be in substantial excess over residual amplification multimerscarried over from the first amplification. The solution containing theamplification oligomers can be transferred to a second solid supportcomprising capture probes with sequences complimentary to a sequence onthe capture extenders. The transferred solution and solid support can bebrought to a temperature above the melting temperatures of theamplification system components, then brought to a stringenthybridization temperature. Many of the hybridization interactions cantake place in solution between the soluble components, but ultimatelythe amplification complex will be captured at the solid support byhybridization of the capture extender to the solid support captureprobes. The solid support can be washed with one or more wash solutions,under stringent conditions. The presence of the amplification oligomer(and associated target nucleic acid in interest in the sample) can bedetected by detection of bound, e.g., fluorescent label probes.

The methods can optionally be used to quantitate the amounts of thenucleic acids of interest present in the sample. For example, in oneclass of embodiments, an intensity of a signal from the label ismeasured and correlated (e.g., through a standard formula determinedthrough regression analysis) with a quantity of the correspondingnucleic acid of interest present. The standard formula can then be usedto calculate an unknown amount of nucleic acid in a sample based on theoutput signal intensity for that sample.

Amplification Oligomers

Amplification oligomers can be the output amplification product of afirst amplification and the input target for a second amplification. Ina typical embodiment, the amplification oligomer is a polynucleotidewith a sequence complimentary to multiple repeat sequences of anamplification multimer and a sequence complimentary to a root sequenceof a label system. In a more preferred embodiment, the amplificationoligomer would also include a sequence complimentary to a capturepolynucleotide sequence. In alternate embodiments, amplificationoligomers comprise a sequence complimentary to multiple repeat sequencesof an amplification multimer, a polymerase primer sequence, and asequence complimentary to a polymerase primer. Other embodiments ofamplification oligomers include a sequence complimentary to multiplerepeat sequences of an amplification multimer and one or more sequencesfunctionally interacting with an amplification system to provide adetectable amplification product.

In a preferred embodiment, an amplification oligomer is a polynucleotidewith a sequence complimentary to the amplification multimer of a firstbDNA amplification, and compliments to label components and (optionally)capture components of a second bDNA amplification. In a more preferredembodiment, the amplification oligomer comprises sequences complimentaryto the multiple M-2 sequences of the amplification multimer of a firstamplification and sequences complimentary to either M-1 sequences of theamplification multimer or L-2 sequences of the label extender of thesecond amplification. In most preferred embodiments, the amplificationoligomer also comprises sequences complimentary to C-2 or C-3 sequencesof second amplification capture components.

In an aspect of the invention, the complimentary sequences of theamplification oligomer have lengths enhancing hybridization reactionkinetics, minimizing stearic interference with desired intermolecularinteractions, and providing melting temperatures compatible with overallsystem requirements. In embodiments described above wherein bothamplification steps are bDNA amplifications, it is preferred that theamplification oligomer sequences complimentary to the M-2 sequencesrange in length from about 20 to about 80 nucleotides, from about 30 toabout 70 nucleotides, or from about 40 to about 60 nucleotides. It ispreferred the amplification oligomer sequences complimentary to the M-1or L-2 sequences range in length from about 15 to 50 nucleotides, fromabout 20 to 40 nucleotides, from 25 to 30 nucleotides, or about 28nucleotides. It is preferred the amplification oligomer sequencescomplimentary to the C-2 or C-3 sequences range in length from about 15to 80 nucleotides, from about 20 to 60 nucleotides, from 25 to 50nucleotides, or about 40 nucleotides. In certain cases, theamplification oligomer sequences complimentary to the M-1 or L-2sequences, or the amplification oligomer sequences complimentary to theC-2 or C-3 sequences include two or more repeat or redundant sequencesso that, e.g., label or capture systems of the second amplification canreadily employ cooperative hybridization features.

The complimentary sequences of the amplification oligomers can be in anyorder along the polynucleotide. It is generally preferred that theamplification oligomer sequences complimentary to the M-2 sequences ofthe first amplification be between the sequences complimentary to theC-2 or C-3 (capture) sequences and sequences complimentary to the M-1 orL-2 (label) sequences. This arrangement can allow better hybridizationkinetics and reduce stearic hindrance, e.g., between the solid supportcomponents and label system. In more preferred embodiments, theamplification oligomer has sequences configured to provide cooperativehybridization. For example, the amplification oligomer can have multiplesequences complimentary to multiple capture or label system sequences.In embodiments with cooperative hybridization, the amplificationoligomer sequences can be complimentary in the order:capture-label-M2-label-capture, label-capture-M2-capture-label,label-capture-M2-label-capture, label-label-M2-capture-capture,M2-label-label-capture-capture, M2-capture-label-label-capture,M2-capture-label-label-capture-label-capture, and the like. The ordercan run 5′ to 3′ or 3′ to 5′, e.g., depending on the orientation ofassociated compliments.

In an aspect of the invention, two or more different amplificationoligomers are provided as amplification products of the firstamplification. In a preferred embodiment, the two or more differentamplification oligomers have sequences complimentary to differentamplification multimer M-2 sequences of the first amplification and/orsequences complimentary to different capture C-2 or C-3 sequences of thesecond amplification. Optionally, the two or more differentamplification oligomers have sequences complimentary to different labelsystem L-2 or M1 sequences. The use of different amplification oligomersequences, e.g., in association with analysis of a single sample canallow multiplexing that can be deconvoluted according to final signalcharacter and/or final signal locations, as discussed below. Forexample, amplification oligomers having different capture complimentscan be directed to capture on solid supports at different physicallocations. Optionally, amplification oligomers with different labelcompliments can associate with, e.g., labels emitting differentfluorescent wavelengths. Optionally, amplification oligomers withdifferent M-2 compliments can be accumulated at different locations(e.g., different beads) in a first amplification so they can be detectedin a standard second amplification separate from other amplificationoligomers accumulated elsewhere in the first amplification.

In another aspect, the melting temperatures of the amplificationoligomer sequences hybridized to their compliments is considered toprovide useful structure/function relationships between components oftwo-stage amplifications. For example, the sequence complimentary to M-2sequences of the first amplification can have a T_(m) within 10° C., 5°C. or 2° C. of other hybridizations (e.g., capture probe/captureextender, capture extender/target. target/label extender, labelextender/amplification multimer) of the first amplification. Provisionof similar T_(m)s can allow more stringent hybridizations and washes,e.g., without excessive background or signal loss. Optionally, thesequence complimentary to M-2 sequences of the first amplification canhave a T_(m) significantly lower than for the other hybridizations, sothat, e.g., the accumulated amplification oligomer can be harvestedduring the melting step without also releasing other amplificationcomponents from the solid support. In such a system, one should considerhybridizing all the target and bDNA amplification components without theamplification oligomer at a higher stringency temperature, thanhybridizing an excess of amplification oligomer at a lower temperaturebefore the wash. In another aspect, it can be preferred that the T_(m)for the M-2 compliment of the first amplification be lower than theT_(m) for the capture compliment or label compliment, e.g., so thatresidual amplification multimers from the first amplification are lesslikely to interfere with a second bDNA amplification.

In other aspects of the invention, the amplification oligomers caninclude a compliment to the amplification multimer of a bDNAamplification, a compliment to the first primer of a PCR primer pair,and a sequence substantially the same as the sequence of the secondprimer of the PCR primer pair. This amplifier oligomer configurationallows a first amplification of a target nucleic acid of interest from acomplex sample that might have enzyme inhibiting materials to produce aproduct more compatible with a second amplification system. For example,the amplified material can be separated from any enzyme inhibitors ofthe sample, e.g., in the wash of the first amplification so provides aconsistent substrate for the PCR second amplification. Amplificationoligomers for a two-stage amplification of bDNA to PCR can have, e.g.,the compliment to the M-2 sequence between the primer sequence andprimer compliment. The PCR can replicate the primers and sequences inbetween in a geometric fashion, as is notoriously well known in the art.The product of the PCR can be detected directly or indirectly byenzymatic or non-enzymatic methods. The amplification oligomers of thebDNA/PCR two-stage amplification can include compliments of capturesequences (e.g., between the PCR primer pair sites) to render the PCRproduct directable to identifiable physical locations.

In an aspect of the invention, amplification oligomers typically do notcomprise a label. The amplification oligomers can be accumulated inamounts that are directly detectable without the need for constitutivelabels. Further, the amplification oligomers are usually not intended tobe detected, identified or quantitated directly, but are intended to actas input target material for a second amplification that provides adetectable labeled product output.

Multiplexing Two-Stage Amplification Signals

Amplification oligomers of the invention are not only a key to two-stageamplifications that substantially enhance the sensitivity of nucleicacid assays, but can also provide ways to perform and read two nucleicacid assays at once for the same sample. In a first bDNA amplification,the hybridization between the target nucleic acid of interest and thelabel system can be specific and unique. That is, a first nucleic acidof interest can hybridize, e.g., through L-2 or M-1 sequences, with afirst amplification multimer, which can specifically accumulate a firstamplification oligomer. At the same time, in the same sample, a secondnucleic acid of interest can hybridize, through, e.g., different L-2 orM-1 sequences, with a second amplification multimer, which canspecifically accumulate a different second amplification oligomer. Ifthe first and second amplification oligomers bind to different solidsupport locations, or hybridize to different labeling systems e.g., in asecond amplification, the presence of the first and second nucleic acidsof interest in the sample can be separately detected at the separatelocations and/or as distinguishable label system signals.

Amplification oligomers can allow the results of two or more nucleicacid analyses to be read at the same time at different locations.Identity information for a target nucleic acid can be retained through achain of specific hybridization. For example, identity of a particularnucleic acid of interest can be retained through specific hybridizationsfrom the nucleic acid to a label extender to an amplification multimerto a to a particular amplification oligomer. The amplification oligomercan have a sequence complimentary to the capture system of a specificsolid support so that amplification oligomer detected at that solidsupport can be read as confirming the presence of the target nucleicacid in the sample. The presence of a second target nucleic acid in thesame sample can be read at a different solid support location determinedby a specific combination of label extenders, amplification multimers,amplification oligomers and solid support capture elements associatedwith the second target nucleic acid.

In an exemplary embodiment of target amplifications multiplexed toseparate solid support locations, a first solid support is provided toallow capture of all nucleic acids of interest that may or may not bepresent in a sample. This solid support can, e.g., non-specifically bindall nucleic acids or, in a preferred embodiment, include capture probeswith a range of capture extenders complimentary to the whole range ofnucleic acids of interest. Captured nucleic acids of interest can thenbe specifically hybridized (directly or through any number of specificextenders) to an array of amplification multipliers. Each of theamplification multimer array members can be specifically hybridized toparticular amplification oligomers each having sequences complimentaryto specific capture elements (e.g., capture probes or capture extenders)bound to defined regions of a second solid support. In a multiplexedtwo-stage amplification, the array of first amplification multimers isspecifically hybridized to their specific target nucleic acids on thefirst solid support. An array of amplification oligomers is provided tospecifically hybridize and accumulate on their complimentaryamplification multimers. When the first solid support is stringentlywashed, amplification multimers and accumulated amplification oligomersnot bound to the solid support through the presence of their targetnucleic acid are removed. A melting solution at elevated temperaturescan then releases bound amplification oligomers from the first solidsupport into the solution. One or more second solid supports can becontacted with the solution of amplification oligomers. In preferredembodiments, the solid support surface includes a matrix of locations(e.g., column/row positions) that each have different specific capturesequences (e.g., capture probe C-2 or capture extender C-3 sequences)capable of hybridizing to one member of the amplification oligomerarray, but not to other members. On contact, under stringenthybridization conditions, the amplification oligomers, previously boundto the first solid support through association with their nucleic acidof interest, segregate to their designated matrix location throughhybridization to their specific capture sequence. At this point in thematrixed two-stage amplification, all the bound amplification oligomerscan be detected through a general non-specific detection—the identityinformation for the target nucleic acids has been retained through theprevious steps and can now be determined as the presence ofamplification oligomers at the specific locations. The amplificationoligomers at each location can all have the same sequence complimentaryto a general use labeling system. For example, all the amplificationoligomers can have a sequence complimentary to the same amplificationmultimer M-1 sequence for binding of the amplification multimer andmultiple associated label probes. Detection of label probe signals atspecific locations of the second solid support can be deconvoluted tospecifically confirm the presence of one or two or more differentnucleic acids of interest in the original sample.

In other embodiments of deconvolution by second solid support location,the second solid support can be a conduit or beads and contact of theamplification oligomer solution to the multiple locations can be inseries or in parallel. For example, the melted solution of amplificationoligomers can be transferred to a chamber containing two or more beads,each bead having a different detectable identification signal and havingcapture systems with different specificity on the surface. Two or moredifferent amplification oligomers can be captured on different beads.After contact with a label system, a label probe signal from a bead witha particular identification signal can confirm the presence of aparticular nucleic acid of interest in the original sample. Alternately,the beads can be lined up in a series and be contacted sequentially withthe melted solution of amplification oligomers for specific capture. Inanother embodiment, the surface of a conduit could be provided with alinear array of different capture components to selectively hybridizeand capture each of the different amplification oligomers at a differentlocation along the conduit. After exposure to a label system, signals atparticular locations along the conduit can confirm the presence ofparticular nucleic acids of interest in the original sample.

In another embodiment of matrixing with bead solid supports, the firstsolid supports are an array of beads having different capture systemsand the identity of the captured target nucleic acids is deconvoluted bysorting the beads. For example, in a first amplification the putativetarget nucleic acids are contacted to an array of beads each having aunique identification signal, and each of which has a different capturesystem with different capture specificity. Any nucleic acids of interestpresent in the sample will hybridize and become specifically bound totheir corresponding bead. All the beads of the array can optionally becontacted with the same general use system of amplification multimersand amplification oligomers. Before, during or after contact with theamplification multimers, the beads can be sorted to separate locationsaccording to their identification signal, e.g., using a particle sorter.The beads can be washed, typically before or during the bead sorting.The second amplification (e.g., of the amplification oligomers bound tothe beads) and detection can take place at the location resulting fromthe sorting process. Detection of second amplification label probesignals at the location or in association with the identity signals ofvarious beads can confirm the presence of one, or two or more, nucleicacids of interest in the original sample.

In still another example of multiplexed two-stage amplification methodsemploying amplification oligomers, each different amplification oligomerhas a unique label system compliment so that different amplificationoligomers at the same location can be separately identified andquantitated. In an exemplary embodiment, two or more target nucleicacids of interest are captured on a first solid support and amplified toaccumulate amplification oligomers specific to those putative targetnucleic acids actually present, as previously discussed above. Theamplification oligomers associated with each target can each havesequences complimentary to different label systems, but can all includesequences complimentary to the same capture system. After washing thesolid support, the accumulated amplification oligomers can betransferred and be captured (specifically or non-specifically) on asecond solid support. The labeling system of the second amplificationincludes an array of amplification multimers, each member of the arrayaccumulating different label probes with distinguishable signals.Different combinations of signals in the second amplification canthereby identify different combinations of target nucleic acids thatwere present in the initial sample. This multiplexed two-stageamplification can provide signals that can be deconvoluted from the samelocation at the same time, e.g., by reading multiple distinguishablesignals. In an optional aspect, the amplification oligomers from thefirst amplification can be captured on the second solid support throughnon-specific chemical interactions. In an optional aspect, afterwashing, the accumulated amplification oligomers can be melted andspecifically or non-specifically captured on the same solid support forthe second amplification. In an optional aspect, the washed accumulatedamplification oligomers, still hybridized to first amplificationmultimers, can take part in the second amplification, e.g., bycontacting with the label system of the second amplification.

In another aspect of two-stage amplifications, the amplificationoligomers associated with different target nucleic acids of interest caneach have compliments to different capture systems and compliments todifferent label systems. In this configuration, the identity of eachnucleic acid can be associated with a particular signal at a particularlocation. For example, a sample containing four different target nucleicacids of interest can be captured on a first solid support (specificallyor non-specifically). The captured nucleic acids can be contacted withfour different amplification multimers that each specifically hybridizewith only one of the target nucleic acids. The amplification multimer“A” hybridizes to the first target nucleic acid and to a firstamplification oligomer, which comprises a compliment to a first labelsystem having a green signal and a compliment to the capture system atlocation (1,1) of a second solid support. The amplification multimer “B”hybridizes to the second target nucleic acid and to a secondamplification oligomer, which comprises a compliment to a second labelsystem having a red signal and a compliment to the capture system atlocation (1,2) of the second solid support. The amplification multimer“C” hybridizes to the third target nucleic acid and to a thirdamplification oligomer, which comprises a compliment to a third labelsystem having a yellow signal and a compliment to the capture system atlocation (1,2) of the second solid support. The amplification multimer“D” hybridizes to the fourth target nucleic acid and to a fourthamplification oligomer, which comprises a compliment to a fourth labelsystem having a yellow signal and a compliment to the capture system atlocation (1,1) of the second solid support. In the first amplificationof the sample, the first, second, third and fourth amplificationoligomers are accumulated on the first, second, third and fourthamplification multimers, respectively. After washing and melting, theamplification oligomers are transferred in the same solution to thesecond solid support where they are captured at locations (1,1), (1,2),(1,2) and (1,1), respectively. In a second amplification comprising thefirst (green), second (red) and third (yellow) amplification systems, apattern of locations and signals can be deconvoluted to identify thenucleic acids of interest in the original sample. That is, the presenceof green and yellow signals at location (1,1) would be identify thepresence of both the first nucleic acid and fourth nucleic acid in thesample. The presence of red and yellow signals at location (1,2) wouldbe identify the presence of both the second nucleic acid and thirdnucleic acid in the sample. Thus, from one sample four nucleic acidswere identified at two locations at the same time. This exemplaryembodiment is not intended to be limiting and one skilled in the artwill appreciate from this description that, depending on the number oflocations and signals employed, the number of nucleic acids identifiableby this two-stage double multiplexed technique can become quite large.For example, about 10⁴ different nucleic acids could be identified fromthe same sample at the same time (e.g., using CCD detection), assumingamplification oligomers in concert with a second amplification havingcombination of 11 different label signals and a second solid supportwith locations in a grid of 30 rows and 30 columns. Alternately, thedetection could be sequential and/or the solid support could include anynumber of beads, e.g., with identity signals.

For an assay to achieve high specificity and sensitivity, it preferablyhas a low background, resulting, e.g., from minimal cross-hybridization.Such low background and minimal cross-hybridization are typicallysubstantially more difficult to achieve in a multiplex assay than asingle-plex assay, because the number of potential nonspecificinteractions are greatly increased in a multiplex assay due to theincreased number of probes used in the assay (e.g., the greater numberof capture extenders and label extenders. In preferred embodiments ofmultiplexed two-stage amplifications, cooperative hybridizations areemployed in the capture and/or labeling interactions. For example, anamplification oligomer in a second amplification can be designed toinclude two different sequences complimentary to two different capture(e.g., C-2 or C-3) sequences. Hybridization stringency can be such thatcapture by only one sequence (or capture by two sequences withsubstantial mis-matches) does not take place. With regard to labelsystems, the amplification oligomer in a second amplification can bedesigned to include two different sequences complimentary to twodifferent label system (e.g., L-2 or M-1) sequences, e.g., for multiplesimultaneous LE-label probe system component interactions. Thisreduction in background through minimization of unintendedcross-hybridization events thus facilitates multiplex detection of thenucleic acids of interest.

Two-Stage Amplification Systems

Amplification systems of the invention include components useful in thepractice of methods of the invention. Typical two-stage amplificationsystems include one or more first solid supports functioning to capturenucleic acids of interest, one or more first amplification multimers, asecond solid support functioning to capture an amplification oligomer, alabel system, and one or more amplification oligomers complimentary tothe first amplification multimer and complimentary to a sequence of thelabel system. The systems can further include hardware and informationsystems, e.g., to facilitate solution handling, process conditioncontrol, signal detection, information storage, and/or the like.

In one aspect, the invention includes, e.g., systems used to practicethe methods herein and/or systems comprising the compositions describedherein. The system can include, e.g., a fluid and/or bead handlingelement, a fluid and/or bead containing element, a laser for exciting afluorescent label and/or fluorescent beads, a detector for detectinglight emissions from a chemiluminescent reaction or fluorescentemissions from a fluorescent label and/or fluorescent beads, and/or arobotic element that moves other components of the system from place toplace as needed (e.g., a multiwell plate handling element). For example,in one class of embodiments, a composition of the invention is containedin a flow cytometer, a Luminex 100™ or HTS™ instrument, a microplatereader, a microarray reader, a luminometer, a colorimeter, or likeinstrument.

The system can optionally include a computer. The computer can includeappropriate software for receiving user instructions, either in the formof user input into a set of parameter fields, e.g., in a GUI, or in theform of preprogrammed instructions, e.g., preprogrammed for a variety ofdifferent specific operations. The software optionally converts theseinstructions to appropriate language for controlling the operation ofcomponents of the system (e.g., for controlling a fluid handlingelement, robotic element and/or laser). The computer can also receivedata from other components of the system, e.g., from a detector, and caninterpret the data, provide it to a user in a human readable format, oruse that data to initiate further operations, in accordance with anyprogramming by the user.

In an exemplary embodiment of the systems, wells 400 of a firstmulti-well plate 401 have a surface presenting a capture probe andholding a first solution containing a combination of first captureextenders, blocking probes, first label extenders, first amplificationmultimers and amplification oligomers. The multiwell plate is in contactwith a heat plate 402 temperature controllable through a computer system403, as shown in FIG. 4. The system includes a second multiwell plate404 with second capture probes on the well surfaces 405, and a secondsolution containing a combination of second capture extenders, secondlabel extenders, second amplification multimers and label probes.

The system components can function together in a two-stageamplification, for example, as follows: 1) a sample containing a nucleicacid of interest is added to the first solution; 2) the computer 403commands the heating plate 402 to provide a melting temperature (e.g.,95° C.) in the wells 400 to melt any hybridized nucleic acids to singlestrand form; 3) the computer 403 commands the heating plate 402 toprovide a stringent hybridization temperature to the solution in thewells 400; 4) components of the first amplification specificallyhybridize with each other to ultimately accumulate a large number ofamplification oligomers at the well surface, as described herein (e.g.,in a complex of capture probes to capture extenders to target nucleicacid to label extenders to amplification multimers to amplificationoligomers); 5) the first solution, with residual components, is removed(e.g., by manual or robotic pipetting); 6) the well is washed, e.g., bymanual or robotic introduction and removal of a wash solutions; 7) thesecond amplification solution is added to the well and the computer 403commands the heating plate 402 to provide a melting temperature to meltthe accumulated amplification oligomers from the well surface into thesolution; 8) the amplification oligomers, now in the secondamplification solution, are transferred to a well of the secondmultiwell plate 404 (held on the heating plate or another heating plate406); 9) the computer 403 commands the heating plate 406 to transitionfrom the melting temperature to a stringent hybridization temperature;10) components of the second amplification specifically hybridize toeach other in a complex to ultimately accumulate a large number of labelprobes at the well surfaces 405, as described herein (e.g., a complex ofcapture probes to capture extenders to amplification oligomers to labelextenders to amplification multimers to label probes); 11) the secondsolution, with residual components, is removed; 12) the well is washed;13) the presence and amount of detectable signal from labeled probes inthe well is detected by a detector system 407; and 14) the detectorsystem 407 transmits signal detection data to the computer system forstorage and evaluation.

First Amplification Systems to Accumulate Amplification Oligomers

In a first amplification, as shown in FIG. 2A, a sample (e.g.,conditioned media or lysed cells) to be analyzed by the system includesa target nucleic acid 214. The target nucleic acid 214 is captured byfirst capture probe 204 on solid support 201 (e.g., a well of amicrotiter plate) through set 211 of synthetic oligonucleotide captureextenders. Each capture extender has a first polynucleotide sequence C-3(252) that can stringently hybridize to the target nucleic acid andsecond polynucleotide sequence C-1 (251) that can stringently hybridizeto the capture probe through sequence C-2 (250) of the capture probe.Typically, two or more capture extenders are used; optionally, onecapture extender can be used to capture a target. Each label extender inlabel extenders set 221 hybridizes to a different sequence on the targetnucleic acid, through sequence L-1 (254) that is complementary to thetarget nucleic acid, and to sequence M-1 (257) on amplification multimer(241), through sequence L-2 (255). Blocking probes (224), whichhybridize to sequences in the target nucleic acid not bound by eithercapture extenders or label extenders, are often used in bDNA assays toreduce non-specific target probe binding. A probe set for a given targetnucleic acid thus consists of capture extenders, label extenders, andoptional blocking probes for the target nucleic acid. The captureextenders, label extenders, and optional blocking probes arecomplementary to non-overlapping sequences in the target nucleic acid,and are typically, but not necessarily, contiguous. In this example, asingle blocking probe is used; typically, an array of different blockingprobes is used in an optimized bDNA assay.

Signal amplification can begin with the binding of the label extendersto the target nucleic acid. The amplification multimer is thenhybridized to the label extenders. The amplification multimer hasmultiple copies of sequence M-2 (258) that is complementary toamplification multimer 241 (it is worth noting that the amplificationmultimer is typically, but not necessarily, a branched-chain nucleicacid; for example, the amplification multimer can include a branched,forked, or comb-like nucleic acid or a linear nucleic acid). Note theamplification of the single initial target nucleic acid 214 captured to,e.g., eighteen amplification oligomers 242 accumulated on the firstamplification multimers.

Second Amplification Systems to Accumulate Label Probes

After removal of the first solution and washing the solid support 201,the accumulated amplification oligomers 242 can be melted from the firstamplification multimers 241 into a second solution containing componentsof the second amplification, e.g., a bDNA amplification system. Theamplification oligomers 242 are captured by second capture probes 264 onsecond solid support 261 (e.g., a well of a microtiter plate) throughsecond set 265 of synthetic oligonucleotide capture extenders. Eachsecond capture extender has a first polynucleotide sequence C-3 (266)that can hybridize to the amplification oligomer 242 and secondpolynucleotide sequence C-1 (267) that can hybridize to the captureprobe through sequence C-2 (268) in the capture probe. Typically, two ormore capture extenders are used in a cooperative hybridization;optionally, one the two capture extenders can have different C-3sequences to capture a target at different positions. Each labelextender in second label extenders set 269 hybridizes to a differentsequence on the target nucleic acid, through sequence L-1 (270) that iscomplementary to the amplification oligomer and to sequence M-1 (271) ofsecond amplification multimer (265), through sequence L-2 (272).Blocking probes (273), which hybridize to sequences in the amplificationoligomer not bound by either capture extenders or label extenders can beused to reduce, e.g., non-specific target/probe binding. A probe set fora given amplification oligomer thus consists of capture extenders, labelextenders, and optional blocking probes for the amplification oligomer.The capture extenders, label extenders, and optional blocking probes arecomplementary to non-overlapping sequences in the amplificationoligomer, and are typically, but not necessarily, contiguous.

Signal amplification can begin with the binding of the label extendersto the amplification oligomer. The amplification multimer is thenhybridized to the label extenders. The amplification multimer hasmultiple copies of sequence M-2 (274) that is complementary to labelprobe 262 (it is worth noting that the second amplification multimer istypically, but not necessarily, a branched-chain nucleic acid). Label275, for example, alkaline phosphatase, is covalently attached to eachlabel probe. (Alternatively, the label can, e.g., be non-covalentlyassociated with the label probes.) After removal of the second solutionand one or more washing steps, labeled complexes can be detected, e.g.,by the alkaline phosphatase-mediated degradation of a chemilumigenicsubstrate, e.g., dioxetane. Luminescence can be reported as relativelight units (RLUs) on a microplate reader. The amount ofchemiluminescence is proportional to the level of target nucleic acidoriginally present in the sample (a relationship describable with astandard function).

Solid Supports

Essentially any suitable solid support can be employed in the methods.For example, the solid support can comprise particles such asmicrospheres (e.g., beads), a conduit surface, or it can comprise asubstantially planar and/or spatially addressable support. Differentnucleic acids are optionally captured on different distinguishablesubsets of particles or at different positions on a spatiallyaddressable solid support. The nucleic acids of interest can be capturedat a solid support by any of a variety of techniques, for example, bybinding directly to the solid support or by binding to a moiety bound tothe support, or through hybridization to another nucleic acid bound tothe solid support. Preferably, the nucleic acids are captured to thesolid support through hybridization with capture probes and/or captureextenders.

In some embodiments of the invention, the solid support has a planarsurface and is typically rigid. The planar surface can be, e.g., thesurface of a slide or an interior surface of a compartment or well.Exemplary materials for the solid support include, but are not limitedto, glass, silicon, silica, quartz, plastic, polystyrene, nylon, ametal, a ceramic, and nitrocellulose. The solid support can, e.g., be amultiwell plate or a glass slide with an array of capture probes laidout in a grid pattern at selected positions.

In embodiments involving assay of a large number of samples in parallel,or multiplexed embodiments wherein many target nucleic acids are assayedfrom the same sample at once, the nucleic acids (e.g., sample nucleicacids of interest or associated amplification oligomers) can be capturedat different positions on a non-particulate, spatially addressable solidsupport. Thus, in one class of embodiments, the solid support comprisestwo or more capture probes, wherein each capture probe is provided at aselected position on the solid support. Two or more subsets of n captureextenders can be provided, wherein n is at least two. Each subset of ncapture extenders can be capable of hybridizing to one of the nucleicacids of interest, and the capture extenders in each subset can becapable of hybridizing to one of the capture probes, thereby associatingeach subset of n capture extenders with a selected position on the solidsupport. Each of the nucleic acids of interest present in the sample canbe hybridized to its corresponding subset of n capture extenders and thesubset of n capture extenders can be hybridized to its correspondingcapture probe, thereby capturing the nucleic acid on the solid supportat the selected position with which the capture extenders areassociated.

Typically, in this class of embodiments, the presence or absence of thelabel at the selected positions on the solid support is detected afterthe second amplification. Since a correlation exists between aparticular position on the support and a particular nucleic acid ofinterest, the presence of a label at a position can indicate thepresence of a particular nucleic acid of interest in the sample.

In another class of embodiments, a pooled population of particlesconstitutes the solid support. The population comprises two or moresubsets of particles, and a plurality of the particles in each subset isdistinguishable (e.g., by a detectable identification signal) from aplurality of the particles in every other subset. Typically,substantially all of the particles in each subset are distinguishablefrom substantially all of the particles in every other subset. Theparticles in each subset typically have associated therewith a differentcapture probe.

Essentially any suitable particles can be used, e.g., particles havingdistinguishable characteristics and to which capture probes can beattached. For example, in one preferred class of embodiments, theparticles are microspheres (e.g., small beads). The microspheres of eachsubset can be distinguishable from those of the other subsets, e.g., onthe basis of their fluorescent emission spectrum, their diameter, or acombination thereof. For example, the microspheres of each subset can belabeled with a unique fluorescent dye or mixture of such dyes, quantumdots with distinguishable emission spectra, and/or the like. As anotherexample, the particles of each subset can be identified by an opticalbarcode, unique to that subset, present on the particles.

Microspheres are preferred particles in certain embodiments describedherein since they are generally stable, are widely available in a rangeof materials, surface chemistries and uniform sizes, and can befluorescently dyed. Luminex Corporation ((www.) luminexcorp.com), forexample, offers 100 sets of uniform diameter polystyrene microspheres.The microspheres of each set are internally labeled with a distinctratio of two fluorophores. A flow cytometer or other suitable instrumentcan thus be used to classify each individual microsphere according toits predefined fluorescent emission ratio. Fluorescently-codedmicrosphere sets are also available from a number of other suppliers,including Radix Biosolutions ((www.) radixbiosolutions.com) and UpstateBiotechnology ((www.) upstatebiotech.com). Alternatively, BD Biosciences((www.) bd.com) and Bangs Laboratories, Inc. ((www.) bangslabs.com)offer microsphere sets distinguishable by a combination of fluorescenceand size. As another example, microspheres can be distinguished on thebasis of size alone, but fewer sets of such microspheres can bemultiplexed in an assay because aggregates of smaller microspheres canbe difficult to distinguish from larger microspheres.

Microspheres with a variety of surface chemistries are commerciallyavailable, from the above suppliers and others (e.g., see additionalsuppliers listed in Kellar and lannone (2002) “Multiplexedmicrosphere-based flow cytometric assays” Experimental Hematology30:1227-1237 and Fitzgerald (2001) “Assays by the score” The Scientist15[11]:25). For example, microspheres with carboxyl, hydrazide ormaleimide groups are available and permit covalent coupling of molecules(e.g., polynucleotide capture probes with free amine, carboxyl,aldehyde, sulfhydryl or other reactive groups) to the microspheres. Asanother example, microspheres with surface avidin or streptavidin areavailable and can bind biotinylated capture probes; similarly,microspheres coated with biotin are available for binding capture probesconjugated to avidin or streptavidin. In addition, services that couplea capture reagent of the customer's choice to microspheres arecommercially available, e.g., from Radix Biosolutions ((www.)radixbiosolutions.com).

Protocols for using such commercially available microspheres (e.g.,methods of covalently coupling polynucleotides to carboxylatedmicrospheres for use as capture probes, methods of blocking reactivesites on the microsphere surface that are not occupied by thepolynucleotides, methods of binding biotinylated polynucleotides toavidin-functionalized microspheres, and the like) are typically suppliedwith the microspheres and are readily utilized and/or adapted by one ofskill In addition, coupling of reagents to microspheres is welldescribed in the literature. For example, see Yang et al. (2001) “BADGE,Beads Array for the Detection of Gene Expression, a high-throughputdiagnostic bioassay” Genome Res. 11:1888-98; Fulton et al. (1997)“Advanced multiplexed analysis with the FlowMetrix™ system” ClinicalChemistry 43:1749-1756; Jones et al. (2002) “Multiplex assay fordetection of strain-specific antibodies against the two variable regionsof the G protein of respiratory syncytial virus” 9:633-638; Camilla etal. (2001) “Flow cytometric microsphere-based immunoassay: Analysis ofsecreted cytokines in whole-blood samples from asthmatics” Clinical andDiagnostic Laboratory Immunology 8:776-784; Martins (2002) “Developmentof internal controls for the Luminex instrument as part of a multiplexedseven-analyte viral respiratory antibody profile” Clinical andDiagnostic Laboratory Immunology 9:41-45; Kellar and lannone (2002)“Multiplexed microsphere-based flow cytometric assays” ExperimentalHematology 30:1227-1237; Oliver et al. (1998) “Multiplexed analysis ofhuman cytokines by use of the FlowMetrix system” Clinical Chemistry44:2057-2060; Gordon and McDade (1997) “Multiplexed quantification ofhuman IgG, IgA, and IgM with the FlowMetrix™ system” Clinical Chemistry43:1799-1801; U.S. Pat. No. 5,981,180 entitled “Multiplexed analysis ofclinical specimens apparatus and methods” to Chandler et al. (Nov. 9,1999); U.S. Pat. No. 6,449,562 entitled “Multiplexed analysis ofclinical specimens apparatus and methods” to Chandler et al. (Sep. 10,2002); and references therein.

Methods of analyzing microsphere populations (e.g. methods ofidentifying microsphere subsets by their size and/or fluorescencecharacteristics, methods of using size to distinguish microsphereaggregates from single uniformly sized microspheres and eliminateaggregates from the analysis, methods of detecting the presence orabsence of a fluorescent label on the microsphere subset, and the like)are also well described in the literature. See, e.g., the abovereferences.

Suitable instruments, software, and the like for analyzing microspherepopulations to distinguish subsets of microspheres and to detect thepresence or absence of a label (e.g., a fluorescently labeled labelprobe) on each subset are commercially available. For example, flowcytometers are widely available, e.g., from Becton-Dickinson ((www.)bd.com) and Beckman Coulter ((www.) beckman.com). Luminex 100™ andLuminex HTS™ systems (which use microfluidics to align the microspheresand two lasers to excite the microspheres and the label) are availablefrom Luminex Corporation ((www.) luminexcorp.com); the similar Bio-Plex™Protein Array System is available from Bio-Rad Laboratories, Inc.((www.) bio-rad.com). A confocal microplate reader suitable formicrosphere and planar matrix analysis, the FMAT™ System 8100, isavailable from Applied Biosystems ((www.) appliedbiosystems.com).

As another example of particles that can be adapted for use in thepresent invention, sets of microbeads that include optical barcodes areavailable from CyVera Corporation ((www.) cyvera.com). The opticalbarcodes are holographically inscribed digital codes that diffract alaser beam incident on the particles, producing an optical signatureunique for each set of microbeads.

The particles optionally have additional desirable characteristics. Forexample, the particles can be magnetic or paramagnetic, which provides aconvenient means for separating the particles from solution, e.g., tosimplify separation of the particles from any materials not bound to theparticles.

Amplification Oligomers

Amplification oligomers of the systems can be essentially as describedin the Amplification Oligomers section of the Methods, above. Theamplification oligomers of the inventive systems typically comprise atleast a sequence complimentary to a first amplification multimer (e.g.,at multiple repeat sequences functioning to accumulate nucleic acids ina non-enzymatic amplification) and one or more sequences complimentaryto components of a second amplification system. With these features, theamplification oligomers can function as the product of a firstnon-enzymatic nucleic acid amplification and as the substrate for asecond enzymatic or non-enzymatic amplification.

Where the second amplification involves a bDNA system, the sequencescomplimentary to components of the second amplification system aretypically complimentary to a second amplification multimer or labelextender (e.g., at one or two sequences functioning to specificallyassociate the amplification oligomer with a label system). Where thesecond amplification system is an enzymatic amplification system, thesequences complimentary to components of a second amplification systemtypically act as a substrate for the enzymes or as a component of thesubstrate, e.g., in association with primer pairs in a PCRamplification.

Amplification oligomers can also provide a means to conserve informationacross process steps of a two-stage amplification. The amount ofamplification oligomer from a first amplification can be correlated toan amount of the associated target nucleic acid present in the originalsample. Amplification oligomer sequences complimentary to capture systemsequences can be used in combination with other system components tophysically sort them to predesignated locations on a second substrateunambiguously associated with a certain putative target nucleic acids.Amplification oligomer sequences complimentary to label system sequencescan be used in combination with other system components to selectpredesignated label probes in a second amplification with apredesignated signal associated with a certain putative nucleic acid.

Capture Systems

The two-stage amplification systems of the invention can employ anynumber of different capture systems to capture nucleic acids of interestor capture amplification oligomers at solid supports in the first orsecond amplification stages, e.g., see the Capturing Nucleic Acidssection, above. Capturing can be specific (e.g., through one or morespecific hybridizations) or non-specific (e.g., through covalentchemistries or non-specific affinities). Capturing can be direct (e.g.,through direct contact between the nucleic acid and the solid supportcapture moiety) or indirect (e.g., through intermediate associations).

In the simplest embodiment, capture systems are solid supportscomprising chemical groups that interact directly and non-specificallywith nucleic acids to be captured. For example, the capturing system canbe nitrocellulose paper. Such capture systems are typically notpreferred, e.g., in a first amplification of a complex sample becausecompetition from nucleic acids not of interest and other samplecomponents can reduce desired capture. Such capture systems aretypically not preferred in many multiplexing embodiments of thetwo-stage amplification system, e.g., because amplification oligomersmay not be directable to specific locations of such solid supports.However, direct non-specific capture solid supports can be useful, e.g.,in capture of accumulated amplification oligomers after a wash step,e.g., for detection of a single nucleic acid of interest or wheremultiplexing is based on signaling from label systems having multiplesignal sets.

In many two-stage amplification systems, the capture system comprises asolid support with little or no affinity for nucleic acids but withattached capture probes for direct or indirect capture of nucleic acidsof interest or capture of amplification oligomers. Direct capture oftarget nucleic acids by capture probes, covalently attached to a solidsupport, has relatively simple kinetics and can provide a good signalwith low background. Direct capture to capture probes avoids the need toengineer and manufacture linking capture extenders. However, directcapture of target nucleic acids with capture probes has the disadvantageof requiring a specially manufactured solid support for each targetnucleic acid of interest.

Capture systems designed with a capture extender to link the targetnucleic acid to the solid support through a generic capture probe offerthe benefit that one solid support can be readily reconfigured forcapture any number of different target nucleic acids by simply providingcapture extenders with capture sequences complimentary to the intendedtarget. Furthermore, a solid support with a generic capture probe can beconfigured to specifically capture two or more targets at once, possiblyat different locations. Such solid supports can also be reusable tocapture different targets in a second use. For example, in a firstamplification, capture extenders with sequences complimentary to thegeneric capture probe and sequences complimentary to a target nucleicacid can indirectly capture the target at the solid support. Anothersolid support, e.g., with the same capture probe can captureamplification oligomers from the first amplification using a secondcapture extender having the sequence complimentary to the capture probeand having a sequence complimentary to the amplification oligomer. Thereis no need to specially manufacture two different solid supports for thefirst and second amplification stages. Systems designed to employ thegeneric capture probes avoid the need to specially manufacture differentsolid supports for different amplification assays directed to differentnucleic acids of interest.

In multiplexed two-stage amplifications wherein deconvolution is basedon the location of a signal, different amplification oligomers aretypically captured at different locations in the capture system. Thedifferent locations can be, e.g., two or more microspheres, differentpositions along a conduit, different matrix positions on a planar solidsupport, different wells of a multi-well plate, and/or the like.Typically, solid supports at the different locations will have captureprobes with different sequences at the different locations. In oneembodiment, a solid support is provided with different capture probes atdifferent locations so that different amplification oligomersaccumulated in a first amplification can be captured directly foramplification and detection at those locations. In another embodiment,the a solid support is provided with different capture probes atdifferent locations to capture capture extenders each combining asequences specific to one capture probe and to one amplificationoligomer, so that the amplification oligomers can be captured indirectlyfor amplification and detection at predesignated locations. In such acase, different sets of capture extenders could be used with a singlegeneric matrixed solid support, e.g., to assay different classes ofamplification oligomers (and associated nucleic acids of interest).

In another multiplexing embodiment, the solid supports comprise sets ofbeads with unique combinations of identification signals and captureprobes. Such beads can be useful in capturing amplification oligomers ina second amplification similar to those described immediately above, butwith deconvolution by correlating a label probe signal to a beadidentification signal instead of a support matrix location. In anotheraspect of bead multiplexing, the beads have unique capture probes forsample nucleic acids of interest and the bead identification signal isused to particle sort the bead to a unique location for the secondamplification and detection. Ultimately, a label signal at the uniquelocation can be correlated to the presence of a particular nucleic acidof interest in the original sample.

Label Systems

Label systems of the present amplification systems are essentially asdescribed above in the Methods section. Label systems in non-enzymatictwo-stage amplifications function to provide a detectable signal in thepresence of one or more captured amplification oligomers. Label systemsinclude a sequence complimentary to an amplification oligomer sequenceand two or more sequences complimentary to a label probe.

Label systems include at least an amplification multimer and labelprobes. Optionally, label systems include one or more label extenders,or the amplification multimer can be complimentary to and hybridizedirectly to the intended amplification oligomer. In a less preferredembodiment for most circumstances, the label system can simply be labelprobes. The amplification multimers can be as described above, e.g., anatural or unnatural nucleic acid comprising multiple sequencescomplimentary to label probe molecules, e.g., a branched DNA, apreamplifier strand associated with an amplifier strand, or a single unbranched amplifier strand.

Typically, the label system sequence complimentary to the amplificationoligomer is a sequence on a label extender or amplification multimer. Inmultiplexing embodiments wherein the presence of two or more differentamplification oligomers is detected as two or more different signals,label systems with different signals are associated with each differentamplification oligomer through unique complimentary sequences. Forexample, two-stage multiplexing amplification systems of the inventioncan include label systems in the second amplification stage with a firstlabel extender complimentary to only a first amplification oligomer andto a first amplification multimer, which has multiple sequence sitescomplimentary to accumulate only those label probes having a firstsignal. The multiplexing amplification system can also have a secondlabel extender complimentary only to a second amplification oligomer andto a second amplification multimer, which has multiple sequence sitescomplimentary to accumulate only those label probes having a seconddistinguishable signal. There can be third, fourth, and fifth signalsets of uniquely associating label extenders, amplification multimersand label probes, and so on. In this regard, a label system can includemultiple uniquely associating sets with different distinguishable labelsignals to detect more than one amplification oligomer (and thereby,uniquely identify the presence of more than one nucleic acid of interestfrom a test sample).

In signal multiplexing embodiments, the label system can include two ormore signal sets. The number of signal sets for signal multiplexing canrange, e.g., from two to 1000, or more; typically from 4 to 20 sets, orabout 10 sets. For example, in label systems using fluorescent labels,present detection technologies readily facilitate detection of several(e.g., 3 to 5) fluorescent probes at the same time. Larger numbers ofsignal sets can be distinguished, e.g., by detecting them separately intime sequence or at different locations. By using label probes withcombinations of signals, the number of uniquely identifiable signal setscan be expanded. For example, label probes that each comprise twodifferent labels can square the number of identifiable label probes fora given number of distinguishable signals.

Labels

Labels associated with label probes can provide the final highlyamplified signal associated with the presence of a nucleic acid ofinterest in a sample. Moreover, labels can be provided with a range ofdistinguishable signals, e.g., useful in multiplexing schemes of theinvention.

A wide variety of labels are well known in the art and can be adapted tothe practice of the present inventions. For example, luminescent labelsand light-scattering labels (e.g., colloidal gold particles) have beendescribed. See, e.g., Csaki et al. (2002) “Gold nanoparticles as novellabel for DNA diagnostics” Expert Rev Mol Diagn 2:187-93.

As another example, a number of fluorescent labels are well known in theart, including but not limited to, hydrophobic fluorophores (e.g.,phycoerythrin, rhodamine, Alexa Fluor 488 and fluorescein), greenfluorescent protein (GFP) and variants thereof (e.g., cyan fluorescentprotein and yellow fluorescent protein), and quantum dots. See e.g., TheHandbook: A Guide to Fluorescent Probes and Labeling Technologies, TenthEdition or Web Edition (2006) from Invitrogen (available on the worldwide web at probes.invitrogen.com/handbook), for descriptions offluorophores emitting at various different wavelengths (including tandemconjugates of fluorophores that can facilitate simultaneous excitationand detection of multiple labeled species). For use of quantum dots aslabels for biomolecules, see e.g., Dubertret et al. (2002) Science298:1759; Nature Biotechnology (2003) 21:41-46; and Nature Biotechnology(2003) 21:47-51.

Labels can be introduced to molecules, e.g. polynucleotides, duringsynthesis or by post-synthetic reactions by techniques established inthe art; for example, kits for fluorescently labeling polynucleotideswith various fluorophores are available from Molecular Probes, Inc.((www.) molecularprobes.com), and fluorophore-containingphosphoramidites for use in nucleic acid synthesis are commerciallyavailable. Similarly, signals from the labels (e.g., absorption byand/or fluorescent emission from a fluorescent label) can be detected byessentially any method known in the art. For example, multicolordetection, detection of FRET, fluorescence polarization, and the like,are well known in the art.

Multiplexing Systems

Multiplexing systems can include, e.g., deconvolution by location ofsignals from a second amplification, by detection of different secondamplification signals at the same location, by detection of three ormore second amplification signals at two locations, or by detection of asecond amplification signal from beads sorted after a firstamplification, as discussed above.

An exemplary embodiment of a two-stage amplification with multiplexingand deconvolution by bead identity is schematically illustrated in FIG.5. In FIGS. 5A to 5C, a first amplification provides an accumulation oftwo different amplification oligomers associated with the presence oftwo different target nucleic acids of interest. FIGS. 5D to 5G describea second amplification on beads wherein the accumulated amplificationoligomers of the first amplification function as substrate on differentbeads. Deconvolution of signals to identify nucleic acids of interest inthe sample can be by correlation of amplification label probe signals toassociated bead identification signals.

FIG. 5A shows solid support 550 with first capture probes 551 havingcaptures three different first capture extenders 552, 553 and 554. Thethree first capture extenders have the same capture probe complimentarysequences, but C-3 sequences complimentary to different sample nucleicacids of interest. As shown in FIG. 5B, different nucleic acids ofinterest 555 and 556 are captured from a test sample, but the nucleicacid complimentary to capture extender 552 was not present in thesample. First label extenders 557 and 558 are complimentary to nucleicacid of interest 555 but not to 556, and complimentary to preamplifiers559 but not to preamplifiers 560. First label extenders 561 and 562 arecomplimentary to nucleic acid of interest 556 but not to 555, andcomplimentary to preamplifiers 560 but not to preamplifiers 559. Asshown in FIG. 5C, preamplifiers 559 specifically hybridize to amplifiers563 and to amplification oligomers 564 but not amplifiers 565 oramplification oligomers 566. With the first amplification configured inthis way: 1) no amplification oligomers are accumulated in associationwith the nucleic acid of interest (not present in the sample)complimentary to capture extender 552, 2) amplification oligomers 564are accumulated in association with nucleic acid of interest 555, and 3)amplification oligomers 566 are accumulated in association with nucleicacid of interest 556 at the solid support 550.

For the second amplification, FIG. 5D illustrates three distinguishablesubsets of microspheres 501, 502, and 503, which have associatedtherewith second capture probes 504, 505, and 506, respectively. Eachcapture probe includes a sequence C-2 (530), which is different fromsubset to subset of microspheres. The three subsets of microspheres arecombined to form pooled population 508, as shown in FIG. 5E. A subset ofcapture extenders is provided for each nucleic acid of interest; subset511 for amplification oligomer 564, subset 512 for amplificationoligomer 567 which is not present, and subset 513 for amplificationoligomer 566. Each capture extender includes sequences C-1 (531,complementary to the respective capture probe's sequence C-2) and C-3(532, complementary to a sequence in the corresponding amplificationoligomer). Three subsets of label extenders (521, 522, and 523 foramplification oligomers 564, 567, and 566, respectively) and threesubsets of blocking probes (524, 525, and 526 for amplificationoligomers 564, 567, and 566, respectively) are also optionally provided.Optionally, the system can employ a single set of label extenderscomplimentary to the same sequence on all amplification oligomers. Eachlabel extender includes sequences L-1 (534, complementary to anamplification oligomer sequence) and L-2 (535, e.g., complementary toamplification multimer M-1 sequences).

Subsets of label extenders 521 and 523 are hybridized to amplificationoligomers 564 and 566, respectively. In addition, amplificationoligomers 564 and 566 are hybridized to their corresponding subset ofcapture extenders (511 and 513, respectively), and the capture extendersare hybridized to the corresponding capture probes (504 and 506,respectively), capturing nucleic acids 564 and 566 on microspheres 501and 503, respectively, as shown in FIG. 5F. Materials not bound to themicrospheres (e.g., capture extenders 512, label extenders 522, andblocking probes 525, etc.) are separated from the microspheres bywashing. Label probe system 540 including amplification multimer 541(which includes sequences M-1 537 and M-2 538), and label probes 542(which contain label 543) is provided. Label extenders 521 and 523 arehybridized to amplification multimers 541, and label probes 542 arehybridized to the amplification multimers, as shown in FIG. 5G.Materials not captured on the microspheres are optionally removed bywashing the microspheres. Microspheres from each subset are identified,e.g., by their fluorescent emission spectrum (i.e., ratio and intensityof λ₂ and λ₃, FIG. 6), and the presence or absence of the label on eachsubset of microspheres is detected (λ₁, FIG. 6). Since each nucleic acidof interest is associated with particular amplification oligomers and adistinct subset of microspheres, the presence of the label on a givensubset of microspheres correlates with the presence of the correspondingnucleic acid in the original sample.

In optional systems, the microspheres described immediately above couldbe replaced with matrix locations on a planar solid support. Like themicrosphere subsets, each location could have a different capture probe.The accumulated amplification oligomers of the first amplification couldbe melted into a solution and transferred to contact the solid supportlocations. After contact with the labeling system and washing, thepresence of one or two or more nucleic acids of interest in the samplecould be determined as the presence of a label probe signal at apredetermined location on the solid support.

The methods are useful for multiplexed detection of nucleic acids,optionally highly multiplexed detection. Thus, the two or more nucleicacids of interest (i.e., the nucleic acids to be detected) optionallycomprise five or more, 10 or more, 20 or more, 30 or more, 40 or more,50 or more, or even 100 or more nucleic acids of interest, while the twoor more subsets of m first capture extenders, solid support locations,second capture extenders or label extenders can comprise five or more,10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or even 100or more subsets.

Solid Support Capture Arrays

An array of capture probes can be prepared on a solid support (e.g., amembrane, a glass or plastic slide, a silicon or quartz chip, a plate,or other spatially addressable solid support), typically with eachcapture probe bound (e.g., electrostatically or covalently bound,directly or via a linker) to the support at a unique selected location.Methods of making, using, and analyzing such arrays (e.g., microarrays)are well known in the art. See, e.g., Baldi et al. (2002) DNAMicroarrays and Gene Expression: From Experiments to Data Analysis andModeling, Cambridge University Press; Beaucage (2001) “Strategies in thepreparation of DNA oligonucleotide arrays for diagnostic applications”Curr Med Chem 8:1213-1244; Schena, ed. (2000) Microarray BiochipTechnology, pp. 19-38, Eaton Publishing; technical note “AgilentSurePrint Technology: Content centered microarray design enabling speedand flexibility” available on the web atchem.agilent.com/temp/rad01539/00039489.pdf; and references therein.Arrays of pre-synthesized polynucleotides can be formed (e.g., printed),for example, using commercially available instruments such as a GMS 417Arrayer (Affymetrix, Santa Clara, Calif.). Alternatively, thepolynucleotides can be synthesized at the selected positions on thesolid support; see, e.g., U.S. Pat. No. 6,852,490 and U.S. Pat. No.6,306,643, each to Gentanlen and Chee entitled “Methods of using anarray of pooled probes in genetic analysis.”

Suitable solid supports are commercially readily available. For example,a variety of membranes (e.g., nylon, PVDF, and nitrocellulose membranes)are commercially available, e.g., from Sigma-Aldrich, Inc. ((www.)sigmaaldrich.com). As another example, surface-modified and pre-coatedslides with a variety of surface chemistries are commercially available,e.g., from TeleChem International ((www.) arrayit.com), Corning, Inc.(Corning, N.Y.), or Greiner Bio-One, Inc. ((www.) greinerbiooneinc.com).For example, silanated and silyated slides with free amino and aldehydegroups, respectively, are available and permit covalent coupling ofmolecules (e.g., polynucleotides with free aldehyde, amine, or otherreactive groups) to the slides. As another example, slides with surfacestreptavidin are available and can bind biotinylated capture probes. Inaddition, services that produce arrays of polynucleotides of thecustomer's choice are commercially available, e.g., from TeleChemInternational ((www.) arrayit.com) and Agilent Technologies (Palo Alto,Calif.).

Suitable instruments, software, and the like for analyzing arrays todistinguish selected positions on the solid support and to detect thepresence or absence of a label (e.g., a fluorescently labeled labelprobe) at each position are commercially available. For example,microarray readers are available, e.g., from Agilent Technologies (PaloAlto, Calif.), Affymetrix (Santa Clara, Calif.), and Zeptosens(Switzerland).

Label Probe Detectors

Label probe products of the second amplification can be detected usingany hardware appropriate to the chosen solid support and label. Wherethe second amplification takes place on a nitrocellulose membrane andthe label is an enzyme, the detector can simply be, e.g., a technicianvisually inspecting the membrane for development of a chromogen. Wherethe solid support is a floor of a well in a multiwell plate and thelabel is a chemiluminescent enzyme, a sequential or parallel formattedplate reader can be appropriate. In embodiments wherein the solidsupport is a bead with an identification signal, the label signal istypically detected using a Fluorometer associated with a flow cytometeror with a charge coupled device viewing the beads settled into a twodimensional array. These exemplary embodiments of appropriate detectorsare not limiting and one skilled in the art would appreciate appropriatevariations.

Kits

Yet another general class of embodiments provides a kit for detectingone or more nucleic acids of interest. In one aspect, the kit includes afirst solid support, a first amplification multimer, and one or moreamplification oligomers. For kits providing a non-enzymatic secondamplification, the kit can further include a second solid support, and alabel system. In certain embodiments, the kit can include appropriatecapture extenders, blocking probes, and/or label extenders. The solidsupports can comprise, e.g., multiwell plates, planar surfaces, planarsurfaces with matrixed arrays, and/or particles. Capture specificity ofsolid support locations can be by, e.g., well position, matrix locationor bead identity signal.

In multiplexed embodiments of kits, many components can be representedwith two or more different subsets. For example, the solid supportparticles can include a population comprising two or more subsets ofparticles, with a plurality of the particles in each subset beingdistinguishable from a plurality of the particles in every other subset.The particles in each subset can have a different set of capture probebonded to the surface. In another aspect, the kit includes a solidsupport comprising two or more capture probes, wherein each captureprobe is provided at a selected position on the solid support.

Example Zip Codes for Multiplexed Assays

The following examples are offered to illustrate, but not to limit theclaimed invention.

The following prophetic example of a multiplexed two-step amplificationshowing how a first amplification can amplify specific amplificationoligomers with unique zip code sequences to generate a highly amplifiedand deconvolutable signal in a second amplification.

In a first parallel amplification (shown in FIG. 7), different targetnucleic acids of interest 70 (gene 1), 80 (gene 2) and 90 (gene 3), arecaptured on capture probes 71, 81 and 91. The target nucleic acidsspecifically hybridize to amplification multimers 72, 82, and 92. Theamplification multimers each specifically hybridize with up to 15amplification oligomers (for purposes of clarity in the figure, only oneis shown per multimer) 73, 83, and 93; thereby amplifying each singletarget nucleic acid to 15 amplification oligomers in the firstamplification.

The amplification oligomers each comprise a 28 base pair compliment (74,84, 94) to a second amplification preamplifier, a 25 to 60 base paircompliment (75, 85, 95) to the replicate amplification multimersequences of the first amplification, and a 50 base pair zip codesequence (76, 86, 96) complimentary to the capture sequences of thesecond amplification. In the present example, the amplification multimercompliment of each different amplification oligomer is not complimentaryto the corresponding sequence on the other amplification oligomers. Inthis way, the presence of each different target nucleic acid in thefirst amplification can result in a specific signal in the secondamplification. That is, in this example the amplification multimercompliment and capture probe compliment (zip code) can function togetherto provide an amplified signal at a particular substrate location, e.g.,a bead surface.

After the amplification of FIG. 7 is washed and melted, theamplification oligomer product of the first amplification is transferredto a second amplification system, as shown in FIG. 8. The secondamplification has beads 100 (only the bead associated with the firstgene is shown here for clarity) each having unique capture probesequences 101 (only one set shown here for clarity) specificallycomplimentary to only one of the zip code sequences of the amplificationoligomer assemblage. In this way, the zip code sequence of theamplification oligomer can function to allow capture only at aparticular identifiable bead surface. For example, the amplificationoligomer 73 associated with the gene 1 target can be uniquely capturedat the zip code sequence 76 by a capture sequence on bead 100 having aunique identifier signal. The preamplifier compliment can hybridize to ageneric preamplifier 102 which in turn hybridizes to 5 generic linearamplifiers 103. Each linear amplifier 103 can hybridize to about 20label probes 104 (for clarity, not all the amplifier length and labelprobes are shown), thus for every amplification oligomer in the secondamplification a signal can be produced from about 400 labels 105 on thebead 100.

Similar second amplifications can take place on other beads, e.g., inthe same solution, with capture sequences specific to zip codecompliments on the other amplification oligomer products of the firstamplification. The beads can have different identification signals sothat generic label signals on specifically identified beads canunambiguously identify the presence of associated target genes in thefirst amplification.

In the two-step amplification of this example, the 15-fold amplificationof target to amplification oligomer and the 400-fold amplification ofthe second amplification of amplification oligomer to label probe resultin an overall 6000-fold amplification from target to label probe.Moreover, the specific sequence function of the amplification oligomers(unique specific amplification multimer compliments and zip codes) allowdeconvolution of detected signals, so that the presence and/or quantityof two or more targets can be measured at the same time from the samesample.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, many of the methods, compositions and apparatusdescribed above can be used in various combinations.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

1-31. (canceled)
 32. An amplification oligomer comprising: a nucleotidesequence complimentary to an amplification multimer M-2 sequence of afirst amplification multimer; and, a nucleotide sequence complimentaryto a label extender L-2 sequence or to an M-1 sequence of a secondamplification multimer.
 33. The amplification oligomer of claim 32,further comprising a nucleotide sequence complimentary to a captureextender C-3 sequence or to a capture probe C-2 sequence.
 34. Theamplification oligomer of claim 33, wherein the nucleotide sequencecomplimentary to the capture extender C-3 sequence or to the captureprobe C-2 sequence comprises from 20 to 60 nucleotides.
 35. Theamplification oligomer of claim 32, wherein the first amplificationmultimer is a component of a first bDNA assay, and the label extenderL-2 sequence or the M-1 sequence of a second amplification multimer arecomponents of a second bDNA assay.
 36. The amplification oligomer ofclaim 32, wherein the nucleotide sequence complimentary to anamplification multimer M-2 comprises from 30 to 60 nucleotides.
 37. Theamplification oligomer of claim 32, wherein the nucleotide sequencecomplimentary to the label extender L-2 sequence or to the amplificationmultimer M-1 sequence of the second amplification multimer comprisesfrom 20 to 40 nucleotides.
 38. The amplification oligomer of claim 32,wherein the nucleotide comprises a total length ranging from 100 to 150nucleotides.
 39. The amplification oligomer of claim 32, wherein thenucleotide sequence complimentary to an amplification multimer M-2sequence of the first amplification multimer; and, the nucleotidesequence complimentary to a label extender L-2 sequence or to anamplification multimer M-1 sequence of the second amplificationmultimer, hybridize to their compliments with Tms within about 5° C. ofeach other.
 40. The amplification oligomer of claim 33, wherein thecapture extender or the capture probe is bound to a particle comprisingan identification signal.
 41. Two or more of the amplification oligomersof claim 33, wherein a first amplification oligomer comprises anucleotide sequence complimentary to a C-3 or C-2 sequence differentfrom a C-3 or C-2 complimentary sequence of a second amplificationoligomer.
 42. The amplification oligomer of claim 33, wherein thecomplimentary sequences are in the following order: 1) the amplificationmultimer M-2 compliment; 2) the capture extender compliment or captureprobe compliment; 3) the label extender compliment or amplification M-1compliment.
 43. The amplification oligomer of claim 33, furthercomprising two or more sequences complimentary to a capture extender C-3sequence or to a capture probe C-2 sequence, thereby providing forcooperative hybridization.
 44. The amplification oligomer of claim 32,further comprising two or more sequences complimentary to the labelextender L-1 sequence or to the amplification multimer M-1 sequence ofthe second amplification multimer.
 45. The amplification oligomer ofclaim 33, wherein the a nucleotide sequence complimentary to anamplification multimer M-2 sequence of a first amplification multimer isbetween: the nucleotide sequence complimentary to a capture extender C-3sequence or to a capture probe C-2 sequence, and the nucleotide sequencecomplimentary to a label extender L-2 sequence or to an amplificationmultimer M-1 sequence of a second amplification multimer. 46-59.(canceled)
 60. A method of identifying amplification oligomers, themethod comprising: providing a first label system comprising a firstsequence complimentary to a first label probe comprising a first label;providing a second label system comprising a second sequencecomplimentary to a second label probe comprising a second labeldifferent from the first label; providing a sample comprising orsuspected of comprising a first amplification oligomer comprising asequence complimentary to a component of the first label system, orwhich sample comprises or is suspected of comprising a secondamplification oligomer comprising a sequence complimentary to acomponent of the second label system; hybridizing the sample with thefirst and second label system; and, detecting a signal from the firstlabel or from the second label; wherein detection of the signal from thefirst label indicates the presence of the first amplification oligomerin the sample or detection of the signal from the second label indicatesthe presence of the second amplification oligomer in the sample.
 61. Themethod of claim 60, wherein the first or second label system comprises:a label probe comprising a sequence complimentary to an amplificationmultimer, which amplification multimer comprises a sequencecomplimentary to a label extender, which label extender comprises asequence complimentary to the first or second amplification oligomer, b)a label probe comprising a sequence complimentary to an amplificationmultimer, which amplification multimer comprises a sequencecomplimentary to the amplification oligomer; or, c) a label probecomprising a sequence complimentary to an amplifier, which amplifiercomprises a sequence complimentary to a preamplifier, which preamplifiercomprises a sequence complimentary to the amplification oligomer. 62.The method of claim 60, wherein the amplification oligomer furthercomprises a sequence complimentary to an amplification multimer M-2sequence.
 63. The method of claim 60, wherein the label systems comprisea label extender functioning to hybridize the first or secondamplification oligomer to the first or second label system,respectively.
 64. The method of claim 60, wherein the first and seconddetectable markers comprise the same type of label but with differentsignals, or wherein the first and second detectable markers comprisedifferent types of labels.
 65. The method of claim 64, wherein the labeltypes are selected from the group consisting of: fluorophores,radionuclides, ligands, enzymes, chromophores, and chemiluminescentcompounds.
 66. The method of claim 60, further comprising identifyingtwo or more amplification oligomers at substantially the same locationby detecting both the first and second labels at the location.
 67. Themethod of claim 66, further comprising identifying the two or moreamplification oligomers at the same time by detecting both the first andsecond labels at substantially the same time.
 68. A method ofidentifying a nucleic acid of interest in a sample, which methodcomprises: providing a sample comprising or suspected of comprising oneor more nucleic acids of interest; providing a first solid support whichcomprises a first capture probe sequence or a first capture extendersequence complimentary to a first nucleic acid of interest; providing asecond solid support which comprises a second capture probe sequence ora second capture extender sequence complimentary to a second nucleicacid of interest; contacting the first and second solid supports withthe sample; capturing the first nucleic acid of interest on the firstsolid support or capturing the second nucleic acid of interest on thesecond solid support; hybridizing a first labeling system directly orindirectly to the first or second nucleic acid of interest; hybridizingthe first labeling system to one or more amplification oligomers;separating the first solid support from the second solid support; and,separately detecting the amplification oligomers that have beenhybridized on each solid support in a nucleic acid assay; whereby thepresence of the first nucleic acid of interest is identified if theamplification oligomers are detected on the first solid support andpresence of the second nucleic acid of interest is identified if theamplification oligomers are detected on the second solid support. 69.The method of claim 68, wherein the label system components comprise: a)a label probe comprising a sequence complimentary to an amplificationmultimer, which amplification multimer comprises a sequencecomplimentary to a label extender, which label extender comprises asequence complimentary to the first or second amplification oligomer, b)a label probe comprising a sequence complimentary to an amplificationmultimer, which amplification multimer comprises a sequencecomplimentary to the amplification oligomer; or, c) a label probecomprising a sequence complimentary to an amplifier, which amplifiercomprises a sequence complimentary to a preamplifier, which preamplifiercomprises a sequence complimentary to the amplification oligomer. 70.The method of claim 68, wherein said separating comprises a separationselected from a group consisting of: particle sorting, cell sorting,magnetic sorting, and separating particles by location.
 71. The methodof claim 68, wherein nucleic acid assay is selected from the groupconsisting of: a bDNA assay, PCR, LCR, a northern blot, a Southern blot,electrophoresis, and light absorbance.
 72. The method of claim 68,wherein the amplification oligomers associated through the labelingsystem with the first nucleic acid of interest are the sameamplification oligomers associated with the second nucleic acid ofinterest.